L. Twenty-four hours later, the cells were treated with different concentrations of BIBR 1532 for 48 hours. At the end of treatment, each well received 10 l of tetrazolium salt and was incubated for 60 minutes at 37 . The formation of formazan dye was assessed using an ELISA reader at 450 nm. a and b represent the dose response curves of MDA-MB 231 and MCF-7 to BIBR 1532, respectively. c represents the effect of GR, while d represents the effect of BIBR 1532 on the mitochondrial metabolism of saos-2 cell line. The O.D. of each condition is a mean of 12 wells. Bars represent values from at least three independent experiments (*P < 0.05, **P < 0.01).Figure 7 demonstrates that 50 M BIBR 1532 did not affect MDA-MB 231 apoptosis except in cells grown in 0 g/l of glucose. Caspase-3 was not expressed in MCF-7 cells, and this result confirmed several reports that indicated caspase-3 deficiency in this cell line. Caspase-3 expression was not detected in the Saos-2 cell line incubated at different glucose concentrations or treated with BIBR 1532. (Data not shown). The results obtained from this experiment suggest that caspase-3 is involved in the induction of apoptosis in BIBR 1532-treated MDA-MB 231 cells that are deprived of glucose. Apoptosis induction was also investigated via annexin V staining. Figure 8 and Tables 1 and 2 shows same results displayed in Figure 7. The percentage of annexin V-positive cells significantly increased in treated cells with BIBR 1532 without glucose compared to the control cells. These data suggest that the growth inhibitory effect of BIBR 1532 on MDA-MB 231 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28381880 cells is partly due to its effect on apoptosis induction under restricted glucose conditions. In the MCF-7 cell line, high concentrations of BIBR 1532 did not elicit apoptosis as observed in MDA-MB 231, unless cells incubated in highglucose concentration (4.5 g/L) are treated with 50 M BIBR 1532, as shown in Figure 8b and Tables 1 and 2.Discussion Due to its major role in cell immortalization, telomerase has become an attractive target for selective cancer therapy. Several approaches have been studied to decrease or completely inhibit the activity of telomerase. Among these efforts, we mention the direct and indirect inhibition of telomerase, immunotherapy using hTERT as a tumorassociated antigen, and gene therapy with a promoterdriven suicide promoter [27]. Because its expression is mainly restricted to tumor cells and it is the rate-limiting subunit in telomerase activity, hTERT has become the main target of inhibition to stop tumor growth. However, these efforts directly face a major obstacle described by the lag phase between the initiation of telomerase inhibition and the impact on the proliferation capacity [29,30]. Therefore, tumor cells with long telomeres will continue to grow upon telomerase inhibition until substantial telomere erosion has occurred. This limitation negatively impacts the application of this inhibition in patients with aWardi et al. Cancer Cell International 2014, 14:60 http://www.cancerci.com/content/14/1/Page 8 ofFigure 6 Effect of telomerase siRNA on cell Hexanoyl-Tyr-Ile-Ahx-NH2 web viability and mitochondrial metabolism. MDA-MB 231 (a) and MCF-7 (b) cells grown in DMEM with different glucose concentrations were equally seeded at a density of 2 ?104 in a 96-well plate 10 min after 20 nM SiRNA transfection. Seventy-two hours later, each well received 10 l of WST-1 and was incubated for 60 minutes at 37 . The formation of formazan dye was assessed using an ELI.
