Vitamin K3 and vitamin C alone or in combination induced apoptosis in leukemia cells by a similar oxidative stress signalling mechanism

Leukemia is a cancer of the bone marrow and blood. The cause of this hematological disorder is currently still unknown. Today, first-line therapy for the treatment of leukemia includes chemotherapy and radiotherapy. Unfortunately, secondary therapy-related acute lymphoblastic leukemia might emerge following chemotherapy and/or radiotherapy for primary malignancies [1]. Therefore, other therapeutic alternatives based on the reactivation of the apoptotic program should be pursued to eliminate cancer cells [2]. Accordingly, the use of vitamin K3 (VK3, also known as menadione [3]) and vitamin C (VC, also known as sodium ascorbate [4]) alone or in combination (VK3: VC [5]) is highly promising in cancer treatment. Yet, the precise pathway(s) by which VK3 and/or VC induce leukemia cell death are not well established. Moreover, given the complexity of death pathways within a cell, placing these pathways in the proper relationship to the drug trigger is challenging.

During the last three decades, vitamin K3 has been known to display anti-tumor action both in vivo and in vitro in human cancer cell lines [4]. Several observations suggest that vitamin K3 might induce apoptosis -a type of cell death- [6] through different biochemical routes including severe depletion of glutathione and sulfhydryl-containing proteins and alteration of intracellular Ca²⁺ homeostasis [7], activation of c-Jun NH₂-terminal kinase (JNK, [8]), activation of Fas/Fas ligand system independently of the pro-apoptotic p53 protein [9], activation of Fas-dependent and Fas-independent pathways [10] and NF-κB activation [11]. Because most studies have looked at a given pathway in isolation using different cell types, the potential interaction between pathways have often not been addressed. Therefore, the complete mechanism(s) of cell death signalization induced by vitamin K3 in a single cell model remains unclear.

Vitamin C is a water-soluble vitamin effective as antioxidant compound under normal conditions [12]. However, Chen and collaborators [13, 14] have shown that pharmacological concentrations of (5-15 mM) vitamin C was pro-oxidant, generating H₂O₂-dependent cytotoxicity toward a variety of cancer cells in vitro and in vivo without adversely affecting normal cells. Interestingly, it has been shown that (10 μM) VK3 in combination of (2 mM) VC kill leukemia cells (e.g., K562 cells) by oxidative stress independent of caspase-3, with a minor percentage of cell displaying mitochondrial depolarisation and DNA fragmentation without chromatin condensation consistent with a necrosis-like cell death [15]. Yet, the mechanism by which vitamin C-induced apoptosis in Jurkat and K562 cells is not yet fully established.

Since it is known that the transcription factor NF-κB [16], p53 [17], c-Jun [18] and caspase-3 [19] are involved in apoptosis signaling, we hypothesized that VK3 and VC might induce cell death in leukemia cells through activation of such factors by oxidative stress. To test this assumption, we sought to investigate the molecular mechanism by which VC, VK3 alone or in combination (ratio 100:1 [20]) induce cell death in Jurkat (clone E06-1) and K562 leukemia cell lines and lymphocyte cells in relation to the aforementioned pro-apoptotic transcription factors and caspase-3. Although, the role of vitamin C as therapeutic compound is still controversial [21], understanding the mechanism of vitamins alone or together may provide insight into more effective anti-cancer therapy.

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 G_o 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 [25] 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 [26]. How H₂O₂ might be involved in cell death process? Takada and co-workers [27] 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 [28]). 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. [12]. However, our results differ from Han et al. [29] 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 [30]. 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 [31]. 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 [16]. 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. [32], our data comply with the notion that p53 is involved in the mechanism of VK3 and VC toxicity in Jurkat and K562 cells [33]. 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. [15], 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 [34]. 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 [35]. 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 [37]. Because cell death is a pure morphological phenomenon [6], 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 [22] compared to other standard methods [38]. 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 [6] rather than autoschizis morphology [37]. 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 [39] and Ogawa et al. [40], altogether our data suggest that VK3 and VC induce apoptosis in leukemia cells by oxidative stress [41]. 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.