br Corresponding authors E mail addresses d
Corresponding authors. E-mail addresses: [email protected] (D. Bashash), shghaﬀ[email protected] (S.H. Ghaﬀari). 1 These two authors contributed equally to this work.
BIBR1532 was noticed in both single treatment strategies (Bashash et al., 2012, 2013a; Kong et al., 2015) and combinational approaches, where the synergistic potential of the inhibitor was shown in combi-nation with chemotherapeutic drugs, such as ATO (Bashash et al., 2013b), doxorubicin (Bashash et al., 2017c), and paclitaxel (Shi et al., 2015). Apart from suppression of telomerase, several reports suggest that BIBR1532 could reduce the survival capacity of malignant cells through telomerase-independent mechanisms (El-Daly et al., 2005; Wardi et al., 2014). El Daly et al. reported that BIBR1532 eﬀectively induced cell death in samples of both CLL and ALL patients with ne-gative telomerase activity; proposing a telomerase-independent me-chanism probably through loss of TRF2 (El-Daly et al., 2005). Another study also suggested that the cytotoxicity of BIBR1532 on BCR/ABL+ leukemic cells is dependent on the presence of functional p53 (Brassat et al., 2011). In consistency with a previous report (Shi et al., 2015), we did not notice any relationship between p53 status and sensitivity to BIBR1532. To the best of our knowledge, to date, no study has ad-dressed the contribution of NF-κB or the role of miRNAs in the response of leukemic cells to BIBR1532. Our results revealed that the alteration of miRNAs expressions upon telomerase inhibition results in induction of apoptosis via suppression of NF-κB in mutant p53-expressing NB4 cells.
2. Materials and methods
A panel of hematologic malignant (Nalm-6, REH, RPMI8226, KMM-1, K562, U937, KG-1, and NB4) and solid tumor (U87, SKBR3, and T4D7) cells were grown in RPMI 1640 medium supplemented with 2 mM L-glutamine, 10% heat inactivated fetal bovine serum, 100 U/ml and 100 µg/ml streptomycin in a humidified 5% CO2 Streptozocin at 37 °C under standard cell culture conditions. Stock solution of BIBR1532 (Boehringer Ingelheim, Biberach, Germany) was made in sterile DMSO. Relevant amounts of BIBR1532 were added into the culture medium to gain the desired concentrations. As negative control, equal volume of DMSO was added in control samples in which the final concentration of DMSO did not exceed more than 0.1% of total volume. r> The inhibitory eﬀect of BIBR1532 on the metabolic activity of the cells was assessed using MTT assay. Briefly, the cells were seeded at the density of 5000/well into a 96-well culture plate and incubated with the inhibitor for 48 h. After removing the media, the cells were further incubated with MTT solution at 37 °C for 4 h. The resulting formazan solubilized with DMSO and the absorption was measured at 570 nm in ELISA reader.
2.3. Trypan blue dye exclusion test of cell viability
To evaluate cytotoxic and anti-proliferative eﬀects of BIBR1532 on acute promyelocytic leukemia (APL) cells, NB4 cells were plated into six-well plates and were incubated in the presence of the diﬀerent concentrations of the inhibitor. After 48 h of treatment, cell suspension was centrifuged and cell pellet was re-suspended in serum-free medium. Afterwards, one part of 0.4% trypan blue (Invitrogen) and one part of cell suspension were mixed and then allowed mixture to incubate 1–2 min at room temperature. The total number of unstained (viable) and stained (nonviable) cells were manually counted and determined. Finally, percentage of viable cells were calculated as follows: Viability (%) = viable cell count / total cell count × 100.
2.4. Telomeric repeat amplification protocol (TRAP assay)
The enzymatic activity of unique reverse transcriptase in response