N‐Nitrosopiperidine and N‐Nitrosodibutylamine induce apoptosis in HepG2 cells via the caspase dependent pathway

Abstract

The human hepatoma cell line (HepG2) exhibited a dose and time-dependent apoptotic response following treatment with N-Nitrosopiperidine (NPIP) and N-Nitrosodibutylamine (NDBA), two recognized human carcinogens. Our results showed a significant apoptotic cell death (95%) after 24h treatment with NDBA (3.5 mM), whereas it was necessary to use high doses of NPIP (45 mM) to obtain a similar percentage of apoptotic cells (86%). In addition, both extrinsic (caspase-8) and intrinsic pathway (caspase-9) could be implicated in the N-Nitrosamines-induced apoptosis. This study also addresses the role of reactive oxygen species (ROS) as intermediates for apoptosis signaling. A significant increase in ROS levels was observed after NPIP treatment, whereas NDBA did not induce ROS. However, N-acetylcysteine (NAC) did not block NPIP-induced apoptosis. All these findings suggest that NPIP and NDBA induce apoptosis in HepG2 cells via a pathway that involves caspases but not ROS.

Antiapoptotic effects of dietary antioxidants towards N‐nitrosopiperidine and N‐nitrosodibutylamine‐induced apoptosis in HL‐60 and HepG2 cells

“…Moreover, alterations in the redox status of the cell to a more oxidizing environment occurs prior to the final phase of caspase activation in many model of apoptosis (Susin et al, 1998;Morel and Barouki, 1999). Thus, the results obtained in our recent works, where NPIP and NDBA can also cause ROS production in human leukemia (HL-60) and human hepatoma (HepG2) cell lines García et al, 2009), were found to be innovative.…”

“…For these experiments, HL-60 and HepG2 cells were cultured in RPMI 1640 Medium and Dulbecco's Modified Eagle's Medium, respectively, without phenol red and without fetal calf serum and subsequently were pre-incubated with vitamin C (5, 10 and 50 μM), DADS or DPDS (1, 2.5 and 5 μM) for 1 h. Significant ROS levels were detected after treatment with 20 and 25 mM NPIP in HL-60 and HepG2 cells, respectively, whereas NDBA (2 mM) only induced ROS production in HL-60 cells García et al, 2009). Consequently, we did not examine the effect of vitamin C, DADS or DPDS on NDBA-induced ROS production in HepG2 cells.…”

“…In combined treatment experiments, we selected N-nitrosamine doses that enhanced the percentage of apoptosis above 50% after 72 h incubation time. HL-60 cells were treated with 20 mM NPIP and 2 mM NDBA for 72 h (74 and 44% apoptotic cells, respectively), whereas it was necessary to use higher concentrations of NPIP (25 mM) and NDBA (2.5 mM) to obtain a similar percentage of apoptosis in HepG2 cells (70 and 51%, respectively) García et al, 2009). Thus, cells were pre-incubated with vitamin C (5, 10 and 50 μM), DADS or DPDS (1, 2.5 and 5 μM) for 24 h. After incubation, HL-60 and HepG2 cells were simultaneously treated with 20 and 25 mM NPIP, respectively, or 2 and 2.5 mM NDBA, respectively, and the corresponding concentrations of vitamin C, DADS or DPDS for 72 h. Briefly, 3 × 10 6 cells were washed with PBS and fixed in 2% formaldehyde in PBS (1 ml) for 1 h at room temperature.…”

“…Consequently, we did not examine the effect of vitamin C, DADS or DPDS on NDBA-induced ROS production in HepG2 cells. Thus, after pre-treatment with vitamin C, DADS or DPDS, 20 mM NPIP or 2 mM NDBA was added for 0.5 h in HL-60 cells and 25 mM NPIP for 1 h in HepG2 cells (García et al, 2009). Then, 2.5 × 10 5 cells were washed with PBS loaded for 30 min with H 2 DCFDA (10 μM) and incubated in a water bath (37°C).…”

“…Several investigations have reported that apoptosis induced by genotoxic carcinogens such as benzo(a)pyrene (Holme et al, 2007), heterocyclic amines (Hashimoto et al, 2004;Shiotani and Ashida, 2004) and Nnitrosamines García et al, 2008a;García et al, 2009) seems to have an important role in cancer development. In our previous studies, a wide variety of methods were employed to characterize the mode of cell death in Nnitrosopiperidine (NPIP) and N-nitrosodibutylamine (NDBA) treated HL-60 and HepG2 cells, such as fluorescence microscopy, flow cytometry analysis (Annexin V/PI, TUNEL assay and specific inhibitors of caspases) and western blot García et al, 2009). The data demonstrated that the inhibition on cell viability by NPIP and NDBA resulted from the induction of apoptosis and not from non-specific cell death or necrosis.…”