and E.B.; formal analysis, W.N.I.W.M.Z.; investigation, W.N.I.W.M.Z.; resources, D.K., J.B. IEC-6, yet both protein staining were recognized in both cells. Lapatinib exhibited cytotoxic properties on ErbB1/ErbB2 expressing cell lines, with intestinal cells becoming more sensitive to lapatinib compared to tumour cells. Lapatinib induced necrosis in tumour cells, while inducing late apoptosis in intestinal cells may clarify lapatinib-induced diarrhoea in individuals administered with the drug which could be due to apoptosis of intestinal epithelial cells leading to barrier disruption and consequently diarrhoea. and mRNA manifestation was determined using Delta CT (2?Ct) method. The experimental threshold (Ct) ideals were calculated by hand by transforming the Ct ideals into relative quantities relative to two housekeeping genes which are and < 0.05. 3. Results 3.1. Lapatinib Inhibited Cell Proliferation in Walker 256 and IEC-6 Walker 256 and IEC-6 were treated with lapatinib at a series of concentrations (1C10 M) to determine the lapatinib dose that could inhibit 50% cell growth (Number 1a). Lapatinib was found to inhibit 50% of Walker 256 rat breast tumour cell growth at 8.40 0.83 M, and at 3.00 0.96 M in the IEC-6 Xanthatin rat jejunum cell collection. Experiments were also carried out with DMSO (lapatinib vehicle), which was assayed in a series of concentrations equivalent to the concentration of lapatinib treatment. DMSO did not cause 50% cell inhibition (Number 1b) at any of the concentrations, which signifies that the vehicle did not influence lapatinib cytotoxic effect on both cell lines. Open in a separate window Number 1 The effect of (a) lapatinib and (b) dimethyl sulfoxide (DMSO) treatment on Walker 256 and IEC-6 cells as assessed by XTT (2,3-= 4). Data offered as mean S.E.M. 3.2. Mechanism of Cell Death Induced by Lapatinib As indicated in the results above, lapatinib was shown to inhibit cell death in both Walker 256 and IEC-6 cells. Therefore, circulation cytometry was carried out to evaluate the mechanism of cell death induced by lapatinib. Percentage of viable, early apoptotic, late apoptotic and necrotic cells in Walker 256 and IEC-6, after treatment with lapatinib at different incubation time were offered in Number 2aCc (Walker 256) and Number 2dCf (IEC-6). At 6 h, lapatinib-treated samples showed a significantly lower quantity of viable cells (58.99 3.21%) (< 0.0001) and higher numbers of early apoptotic cells (24.71 1.39%) (< 0.0001), compared to control untreated (viable cells: 79.97 0.99%, early apoptotic cells: 7.30 2.51%) (Number 2a), as determined by flow cytometry. However, lapatinib-treated samples did not display any difference in the percentage of viable, early apoptotic, late apoptotic and necrotic cells at 24 h incubation (Number 2b) compared to control untreated Xanthatin samples (> 0.05), while at 48 h incubation, lapatinib-treated samples were shown to have a lower percentage of viable cells (50.70 7.27%) (< 0.05) and higher percentage of necrotic cells (37.91 7.08%) (< 0.01), compared to Xanthatin control untreated samples (viable cells: 71.93 6.71%, necrotic cells: 11.86 5.62%) (Number 2c). Open Xanthatin in a separate window Number 2 The percentage of viable, early apoptotic, late apoptotic and necrotic cells in lapatinib-treated Walker 256 cells compared to control untreated at (a) 6 h SAT1 (b) 24 h (c) 48 h incubation and lapatinib-treated IEC-6 cells compared to control untreated at (d) 6 h (e) 24 h (f) 48 h incubation as quantified via FACS analysis. Graph shown for each cell line is definitely representative of experiments conducted. Results shown within the graph are offered as imply S.E.M (= 6). Results were compared with control untreated cells at the same incubation time in the same category. Data showing the characters were significantly different at the level of < 0.05. Xanthatin a for < 0.05 compared to control untreated cells, b for < 0.01 compared to control untreated cells, A for < 0.0001 compared to control untreated cells. As for IEC-6, the results did not display any significant variations in cell viability at 6 h incubation (> 0.05) (Figure 2d). However, lapatinib-treated samples at 24 h incubation.