In the present study, we provide for the first time in vitro evidence supporting a causative role for oxidative stress in VK3-, and VC-induced apoptosis in Jurkat and K562 cells in a domino-like mechanism involving O2
.-/H2O2, mitochondrial depolarization, transcription factor activation such as NF-κ, p53, and c-Jun converging in caspase-3 activation and apoptotic morphology. Most importantly, it is shown that high concentration of VC (e.g. 10 mM) alone or in combination with VK3 (e.g. 10 μM) in a ratio 1000:1 and 100:1 induced apoptosis in Jurkat and K562 cells, respectively by a comparable mechanism to VK3 (e.g. 10 μM) and VC (e.g. 10 mM) alone. Moreover, by using antioxidant compounds, we demonstrated that O2
.-/H2O2 production are essential in VK3-and VC-induced cytotoxicity. This notion was further reinforced by the fact that the antioxidant CP55,940 and NAC completely protect leukemia cells against VK3- and VC-superoxide radicals and H2O2 toxicity. Most importantly, lymphocytes were more resistant to cell death induced either by VC or VK3 treatment alone than leukemia cells. We speculate that differences in cell cycle (i.e., lymphocytes are in Go cell cycle and cancer cells are constantly dividing cells), differences in glutathione content and/or differences in gene expression of antioxidant proteins (e.g., catalase, thioredoxin, superoxide dismutase, glutathione peroxidase) may explain resistance and/or vulnerability in lymphocyte and leukemia cells against VK3 and VC exposure. Taken together our results suggest that different compounds with varied chemical properties might present a similar oxidative stress mechanism of action to erode leukemia cells.
We confirm that VK3  and VC at high concentrations [13, 14, 26] generate O2
.- and H2O2. Moreover, by using antioxidant compounds, it is shown that both reactive oxygen species are related to apoptotic morphology. Our result comply with the notion that H2O2, as a by-product of O2
.- dismutation, provokes apoptosis in Jurkat and K562 cells . How H2O2 might be involved in cell death process? Takada and co-workers  have demonstrated that H2O2 induces NF-κB activation in Jurkat cells through the spleen tyrosine kinase (Syk). Alternatively, H2O2 may activate NF-κB through phosphorylation IκBα kinase (IKKα) and IKKβ ). In accordance with these observations, we found p65-DAB+ nuclei by immunohistochemistry technique in both cell lines treated with either VK3 or VC, as manifestation of p65 activation and translocation to the nuclei, but almost undetectable in untreated cells. Moreover, pharmacological inhibition of NF-κB with PDTC inhibited to almost control value the apoptotic morphology. Taken together these data suggest that VK3 and VC induce activation and translocation of the NF-κB (p65) probably via H2O2-induced mechanisms. Our results are in agreement with Jones et al. . However, our results differ from Han et al.  who showed VC toxicity in HL-60 cells through down-regulation of NF-κB activity. Although the nature of this discrepancy is not known, one possible explanation is that HL-60 cells constitutively express NF-κB (also called "NF-κB positive" cancer cells), which confers cell survival . We conclude that depending on the inducible or constitutive expression NF-κB profile in cells, NF-κB will be an important factor to determine the appropriate therapeutic strategy against lymphoblastic/myelogenous leukemia. Consequently, NF-κB may constitute an important therapeutic target . In accordance with other scientific reports and our present data suggest that activation of NF-κB is linked to cell death signaling in Jurkat and K562 cells . Indeed, NF-κB transcribes pro-apoptotic genes such as p53. Immunohistochemical detection of p53 may indicate that p53 can be directly up-regulated by NF-κB. Moreover, pharmacological inhibition of p53 by PFT completely abolishes VK3 and VC-evoked apoptosis. In contrast to Karczewski et al. , our data comply with the notion that p53 is involved in the mechanism of VK3 and VC toxicity in Jurkat and K562 cells . It is noteworthy to mention that p53 transcribes pro-apoptotic genes such as PUMA, Noxa, Bim, Bid, Bax, which are able to permeabilize mitochondria, thus promoting the release of the apoptogenic cytochrome c, which elicits caspase-3 protease activation, leading to nuclear chromatin fragmentation, typical of apoptotic morphology. In fact, caspase-3 NSCI inhibitor protected cells from those noxious stimuli. In contrast to Verrax et al. , our data clearly showed that VC, VK3 or VC/VK3 induced toxicity in Jurkat and K562 cells by caspase-3 dependent form of cell death.
According to immunohistochemical staining and pharmacological blockade, we found that VK3 and VC activate c-Jun. How, then, these concurrent signaling pathways are activated either by VK3, VC or H2O2? As mentioned above, H2O2 can directly activate NF-κB through Syk or indirectly by activation of the multisubunit IKKα/β by Syk or MEKK1/MAPKK kinase. Therefore, one possibility is that once MEKK1 is activated, it may serve as a cross talk molecule between the JNK and NF-κB pathway . Taken together our findings and this information, it is reasonable to assume that NF-κB and JNK/c-Jun are accomplices with each other during VK3/VC-induced apoptosis in leukemia cells. Furthermore, JNK interacts with p53 in response to stress . We conclude that induction of NF-κB, JNK/c-Jun, and p53 by VK3 and VC might be particularly suitable for the treatment of leukemia [31, 36].
During the last decade, VC and VK3 administered in a ratio 100:1 respectively, exhibit synergistic anti-tumor activity by a cell death process denominated autoschizis. This cell death is a type of necrosis characterized by exaggerated membrane damage and progressive loss of cytoplasm through a series of self-excisions . Because cell death is a pure morphological phenomenon , we used acridine orange/ethidium bromide (AO/EB) staining as one of the most reliable and unbiased method to identify live, (early and late) apoptotic and necrotic cell  compared to other standard methods . Therefore, we first consistently found that (10 μM) VK3 and (10 mM) VC induced (early and late) apoptosis judged against N,N,N′,N′-tetrakis-(2-Pyridylmethyl)ethylenediamine (5 μM, TPEN) reagent, which induces 100% nuclei apoptotic morphology in a caspase-3 and p53-dependent fashion in Jurkat cell line (control, data not shown). We found a ratio 100:1 VC: VK3 induced no cell death and mitochondrial damage in Jurkat cells. However, a significant percentage of (late) apoptotic morphology and depolarized mitochondria in cells were provoked by a ratio 1000:1 (VC: VK3) compared to cells treated with VC and VK3 alone. In agreement with others data, it is found that a ratio 100:1 VC: VK3 induced cell death and mitochondrial damage in K562. Strikingly, Jurkat and K562 cells showed classical apoptosis and necrosis  rather than autoschizis morphology . Furthermore, it was found that (1000:1; 100:1) VC/VK3 activates NF-κB, p53, c-Jun and caspase-3, typical of apoptosis. In agreement with Sakagami et al  and Ogawa et al. , altogether our data suggest that VK3 and VC induce apoptosis in leukemia cells by oxidative stress . In contrast to other reports [15, 42–44], our data suggest that autoschizis might not be operative, at least under the present experimental conditions, in Jurkat and K562 cells. Taken together these results imply that VC- and VK3-induced autoschizis is a cell-specific rather than universal cell death process. Altogether our data suggest that a (1000:1; 100:1) VC: VK3 dose should be an intravenously therapeutic dose in the treatment of lymphoblastic and myelogenous leukemia.