Carcinoma

Example 5

In order to compare levels of adherence to HEp-2 epithelial cells in culture, we used an established model for evaluating adherence of EHEC O157:H7 (27). HEp-2, human laryngeal carcinoma epithelial cells, were a kind gift from Dr. Carlton Gyles (Department of Pathobiology, University of Guelph). Briefly, HEp-2 cells grown in EMEM supplemented with 10% (v/v) FBS were plated onto 24-well tissue culture plates at 2×10⁵ cells ml⁻¹ and incubated for 24 h in the presence of 5% CO₂. The cells were then maintained during the assay in serum and antibiotic-free EMEM. Before inoculation with bacteria, 10% (v/v) of L. acidophilus CFSM selected fractions were added in triplicate to treatment group wells. Wells containing the negative control groups were inoculated with 10⁵ E. coli O157:H7 strain VS94 with or without supplementation with 100 μM propanolol (Sigma-Aldrich Canada Ltd.). Following inoculation of 10⁵ EHEC O157 into treatment and control group wells, the plates were incubated for 3 h at 37° C. in the presence of 5% CO₂. The cell monolayers were then washed three times with PBS to remove non-adhering bacteria and fresh medium was added. Cells were incubated for another 3 h and then washed six times with PBS. Washed cells were lysed with 0.1% Triton X-100. Released bacteria present in the suspension were collected and appropriate dilutions were plated on LB agar. To evaluate if the percentage of adherence in the treatment groups was significantly different from that of the control group, where the recovered counts from the control group (2.2×10⁷ CFU ml⁻¹) were considered to be 100%, the percentage of adherence in the negative control and treatment groups were calculated using the following equation.

$\%\mathrm{of}\mathrm{Recovery}=\underset{2.2\times {10}^7}{\underset{\_}{\mathrm{Group}}}\mathrm{CFU}{\mathrm{ml}}^{-1}\underset{\_}{\times 100}$

Example 5

The human skin squamous carcinoma line (HSC) (Japanese Collection of Research Bioresources Cell Bank (JCRB)) was cultured in Dulbecco's minimal essential media (DMEM) containing 20% fetal bovine serum (FBS), (Thermofisher, Waltham, MA). Cells were plated at a density of 5,000 cells per well into a 96-well plate and allowed to attach for 24 h in a 37° C. humidified incubator with a 5% CO₂ atmosphere. Media was replaced with that containing test agents or vehicle (0.1% dimethylsulphoxide) in 1% FBS and cells incubated for a further 72 h. Cell viability was assessed using CellTiter-Glo® (Promega). Non-linear regression analysis was performed using GraphPad PRISM®. FIG. 5 shows a representative experiment where the EC₅₀ of COMPOUND A, was 5.2 nM and that of COMPOUND G was 9.2 nM.