Handbook of Vitamin C Research

116Ana I. Haza, Almudena García and Paloma Moralesto obtain more insight into the vitamin C effect, we explored the feasible induction ofapoptosis using the TUNEL assay. Our results showed that the viability of HepG2 cells wasnot altered by supplementation of the medium with vitamin C at any of the concentrationsand times tested (Figure 12 A and 13 A). Lazarová and Slamenová [82] observed that 0.5mM vitamin C did not induce any cytotoxic effect on HepG2 cells. However, our resultsreveal that there were significant losses of cell viability, measured by MTT assay, 72 h aftertreatment of HL-60 cells with 50 and 100 μM vitamin C (29 and 46%, respectively) (Figure12 B). Also, LDH assay demonstrated that 50 μM at 72 h and 100 μM vitamin C at 48 and 72h were, in fact, toxic to HL-60 cells, since LDH release was increased up to 36% (Figure 13B). These findings are in agreement with Blasiak et al. [104] and Bhat et al. [105] whoreported that concentrations of vitamin C ranging from 20 to 200 μM are able to causeoxidative DNA breakage in human lymphocytes. Moreover, the percentage of apoptotic cellsfound in TUNEL assay increased to 21% in HL-60 cells when they were treated with 100 μMvitamin C for 72 h (Figure 14). Previous reports have revealed that vitamin C is selectivelytoxic to some types of tumor cells [106]. Park et al. [107] showed that vitamin C (0.25-1 mM)induced apoptosis in leukemic cells (HL-60 and NB4), whereas the same concentrations ofvitamin C had no significant effect on three ovarian cancer cell lines (SK-OV-3, OVCAR-3and 2274).The potential anti-apoptotic of vitamin C by its antioxidant capacity was previouslyshown in human leukemia (HL-60) [108] and colon carcinoma (HT-29) cells [109]. VitaminC (50 M) exerted similar protective effect in HL-60 and HepG2 cells towards NPYR (65%and 63% of reduction, respectively) (Figure 15 B) and NPIP (77% and 74% of reduction,respectively) (Figure 15 C), two cyclic N-Nitrosamines. However, the inhibitor effect ofvitamin C (50 M) towards apoptosis induced by lineal chain N-Nitrosamines, such asNDMA (Figure 15 A) and NDBA (Figure 15 D), was higher in HL-60 cells (75% and 80% ofreduction, respectively) than in HepG2 cells (57% and 66% of reduction, respectively). Thus,a possible explanation of the variation in the anti-apoptotic effect of vitamin C could beattributed to the differences in the levels of enzymatic activities in HepG2 and HL-60 cells.We have previously reported that NPIP and NDBA induced a significant ROS productionin HL-60 cells [44], whereas ROS levels were only enhanced by NPIP in HepG2 cells, butnot by NDBA [45]. Therefore, the scavenging ability of vitamin C was evaluated towardsROS production induced by NPIP in both cell lines and by NDBA in HL-60 cells. It was notdetected significant ROS levels after pre-treatment with only vitamin C (5-50 M) (Figure16). In combined treatment experiments, we have observed that vitamin C decreased the ROSproduction induced by NPIP and NDBA to baseline levels. Accordingly, the inhibition ofNPIP and NDBA-induced apoptosis by vitamin C could be related to its ROS trappingability.The two major mechanisms regulating apoptosis include the extrinsic pathway triggeredby death receptors and the intrinsic pathway mediated by mitochondrial events. The apicalproteases in the extrinsic and intrinsic pathways are caspase-8 and caspase-9, respectively.Recently, we have showed that in NPYR and NDMA-induced apoptosis mainly operates thecaspase-8-dependent pathway, but there is also a caspase-9-dependent side pathway [46].However, NPIP and NDBA-induced apoptosis in HepG2 cells was through the activation ofboth the extrinsic and the intrinsic pathway [45]. Perez-Cruz et al. [110] found that vitamin C