Abstract
Background/Aim: This study analysed the effect of α-tocopheryl succinate (α-TS) on the redox-state of leukemia and normal lymphocytes, as well as their sensitization to fifteen anticancer drugs. Materials and Methods: Cell viability was analyzed by trypan blue staining and automated counting of live and dead cells. Apoptosis was analyzed by FITC-Annexin V test. Oxidative stress was evaluated by the intracellular levels of reactive oxygen species (ROS) and protein-carbonyl products. Results: Most combinations (α-TS plus anticancer drug) exerted additive or antagonistic effects on the proliferation and viability of leukemia lymphocytes. α-TS combined with barasertib, bortezomib or lonafarnib showed a strong synergistic cytotoxic effect, which was best expressed in the case of barasestib. It was accompanied by impressive induction of apoptosis and increased production of ROS, but insignificant changes in protein-carbonyl levels. α-TS plus barasertib did not alter the viability and did not induce oxidative stress and apoptosis in normal lymphocytes. Conclusion: α-TS could be a promising adjuvant in second-line anticancer therapy, particularly in acute lymphoblastic leukemia, to reduce the therapeutic doses of barasertib, bortezomib, and lonafarnib, increasing their effectiveness and minimizing their side effects.
The application of natural substances as adjuvants in second line cancer therapy has always attracted the attention of researchers and clinicians. Many natural products such as ascorbic acid, polyphenols, melatonin, docosahexaenoic acid, quinones, and others are characterized by anticancer activity (1-32). Unlike conventional chemotherapeutics, some natural products can alter intracellular redox homeostasis and induce apoptosis in cancer cells without causing toxic side effects on normal cells and tissues (4). Such natural redox modulators can activate unique biochemical pathways specific to cancer cells that lead to suppression of proliferation, decreased viability and/or cell death. Our previous studies also confirmed the cytotoxicity of some natural redox modulators to leukemic lymphocytes, while decreasing the harmful side effects of conventional chemotherapeutics to normal lymphocytes (5-9).
Alpha-tocopheryl succinate (α-TS), an ester of α-tocopherol (the main ingredient of vitamin E), is a biologically active lipophilic compound with redox modulating properties (10, 11). Recent studies have demonstrated the possibility of using α-TS in drug delivery systems due to its low molecular weight and the existence of an intracellular membrane transport, as well as the possibility of self-assembly in nanocarriers (12-15). α-TS can be also incorporated into polymeric hydrophobic vesicular membranes using a simple nano-engineered dialysis method, whereby multiple non-covalent interactions are formed (14). In this context, α-TS has attracted the attention of pharmacists over the last decade.
α-TS is considered the most effective form of vitamin E compared to α-tocopherol, α-tocopheryl acetate and α-tocopheryl nicotine, to induce differentiation, inhibit proliferation and induce apoptosis in cancer cells, depending on its concentration (15). Various studies have shown selectivity of α-TS for different types of cancer cells with respect to induction of cell death (10, 14-16), without affecting the viability of normal cells (17). The anticancer effects of α-TS have also been reported in several experimental cancer models, including animals with breast (18), lung (19), prostate (20), and colon cancer (21), as α-TS is administered intraperitoneally or intravenously to avoid the hydrolysis in the gastrointestinal tract due to the enzymatic activity of intestinal esterases (22).
The exact mechanism by which α-TS manifests its specific effect on the viability and/or proliferative activity of cancer cells is not yet fully understood, but its role in impairing cancerous mitochondria and altering the redox state of cancer cells have been described (23-36). Studies have shown that α-TS alters the redox homeostasis of cancer cells and provokes the generation of reactive oxygen species (ROS) (23, 24, 26, 27), although α-tocopherol – the main product of de-esterification of α-TS, is one of the strongest antioxidants. For example, dos Santos et al. have found that α-TS induces ROS production in NB4 cells (derived from acute promyelocytic leukemia) hours before the expression of typical apoptotic markers, such as cytochrome c release from mitochondria and caspase activation (24). Superoxide dismutase and catalase had no effect on α-TS-induced ROS generation. In addition, α-TS caused a progressive reduction in the levels of glutathione in these leukemia cells. The authors suggested that α-TS may directly affect the function of the mitochondrial electron transport chain, especially complex I, due to its reaction with glutamate/malate, accompanied by depletion of NADH levels and subsequent reduction of oxygen consumption in mitochondria (24). The effect of α-TS on ROS production and cellular respiration is also explained by the inhibition of succinate-binding site of complex II and the proximal CoQ10 binding site of the electron transport chain (24, 26). These mechanisms may underlie the targeting effects of α-TS on cancer cells (26).
Acute lymphoblastic leukemia is the most common cancer in children and the disease is more aggressive in infants and those over 10 years of age. Despite a significant reduction in the risk of relapse and a corresponding increase in overall survival, it is considered that the conventional chemotherapy has reached its limits regarding efficacy and tolerance (28). Conventional anti-leukemia drugs can also attack normal immune cells as well as other non-cancerous dividing cells of the growing child’s organism. In this context, it is important to consider potentiating conventional chemotherapy with carefully selected substances that specifically sensitize leukemia cells and/or activate the immune system (29). In this regard, combination therapy involving two or more drugs with different molecular mechanisms has been described as an effective alternative approach in the treatment of leukemia (14).
The present study aimed to investigate the effect of α-TS on the redox-state of leukemia and normal lymphocytes, as well as their sensitization to four conventional and eleven new-generation anticancer drugs. We also discuss the molecular mechanisms of action of α-TS (in combination with anticancer drugs), based on the regulation of metabolic processes that are vital to leukemia lymphocytes but not to normal lymphocytes.
Materials and Methods
Cells and treatment protocol. The experiments were performed on: (i) leukemia lymphocytes (Jurkat), derived from patients with acute lymphoblastic leukemia and (ii) on normal lymphocytes, isolated from healthy blood donors. The cells were cultured in RPMI-1640 medium (Sigma-Aldrich, Weinheim, Germany), supplemented with 10% heat-inactivated fetal bovine serum (Gibson, Nashville, TN, USA and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin) (Gibson) in a humidified atmosphere at 37°C with 5% CO2. All cells were collected by centrifugation (1000 × g, 10 min). They were refed with a fresh medium without antibiotics before treatment with anticancer drugs.
The drugs were dissolved in culture grade dimethyl sulfoxide (DMSO; Sigma-Aldrich, Weinheim, Germany) or phosphate-buffered saline (PBS; 10 mM, pH 7.4). The final concentration of DMSO in the cell suspension did not exceed 1% to avoid influencing cell viability.
The cells (1×106 cells/ml) were incubated with α-TS, drug, or drug plus α-TS at the following concentrations: α-TS (25 μM), ABT-737 (0.1 μM), AZD-7762 (0.1 μM), μM BEZ-235 (0.025), barasertib (0.05 μM or 0.01 μM), bleomycin (0.5 μM), bortezomib (0.01 μM), cisplatin (2.5 μM), doxorubicin (0.1 μM), everolimus (5 μM), lomustine (10 μM), lonafarnib (0.5 μM), MG-132 (0.025 μM), MLN-2238 (0.01 μM), PI-103 (0.5 μM), palbociclib (0.25 μM).
α-TS, AZD-7762, doxorubicin, and lomustine were purchased from Signa-Aldrich. ABT-737, BEZ-235, barasertib, bortezomib, cisplatin, PI-103, everolimus, lonafarnib, MLN-2238, palbociclib were obtained from Selleckchem (Huston, TX, USA). Bleomycin was obtained from Nippon Kayaku Co. (Tokyo, Japan) and MG-132 from Wako (Tokyo, Japan).
Cells were incubated with α-TS, the drug or their combination (α-TS + drug) for different time intervals. At each time interval, aliquots were used for cell viability assay. The selected concentrations of the drugs and α-TS applied separately induced approximately 20-30% inhibition of the growth of leukemia lymphocytes.
Cell viability assay. Cell viability was analyzed using trypan blue staining and Countess™ Automated Cell Counter (Invitrogen, Oregon, USA).
Briefly, 10 μl of trypan blue (0.4%) were added to 10 μl of cell suspension and incubated for 30 s, and 10 μl of the cell suspension was placed in a Countess® glass chamber (Invitrogen). The number of live and dead cells in the suspension was counted automatically. The linear range to operate the automated cell counter was 1×104–5×106 cells/ml, and the optimal cell size was in the range of 5-60 μm.
Intracellular ROS assay. The amount of ROS was analyzed using OxiSelect™ In vitro ROS/RNS Assay Kit – Green Flourescence (Cell Biolabs Inc., San Diego, CA, USA), following the manufacturer’s instruction. 2’,7’-Dichlorodihydrofluorescein (DCF) was used as a standard. The amount of DCF product, obtained due to interaction between ROS and the fluorogenic probe 2’,7’-dichloro-dihydrofluorescin DiOxyQ (DCFH-DiOxyQ) was analyzed spectrofluorimetrically at λex=480 nm and λem=530 nm, using Tecan Infinite F200 PRO (Tecan Austria GmbH, Mannedorf, Austria) microplate reader. Details are described in our previous articles (7, 8).
Protein–carbonyl assay. The level of oxidative stress in biological samples was also analyzed by the amount of protein–carbonyl derivatives of prolin, arginite, lysine, and threonine, using OxiSelect™ Protein Carbonyl Spectrophotometric Assay Kit (Cell Biolabs Inc.). The analysis is based on derivatization of the carbonyl groups with dinitrophenylhidrazine with formation of protein– hydrazone, which is detected spectrophotometrically at 375 nm. Oxidized bovine serum albumin was used as a standard. Details are described in our previous articles (7, 8).
Apoptosis assay. The induction of apoptosis was analyzed, using FITC-Annexin V Apoptosis Detection Kit (BioVision, Milpitas, CA, USA), as it was described in the manufacturer’s instruction. The analysis is based on the detection of phosphatidylserine (PSer) expression on the surface of apoptotic cells. FITC-annexin V bound to PSer exposed on the cell surface was detected spectrofluorimetrically at λex=488 nm and λem=535 nm, using a Tecan Infinite F200 PRO (Tecan Austria GmbH) microplate reader. Details are described in our previous articles (7, 8).
Results
Effect of α-TS on the viability of leukemia and normal lymphocytes. To select the optimal concentration of α-TS (IC20) required for its combination with conventional drugs, we treated both leukemia and normal lymphocytes with different concentrations of this redox modulator in the range of 25 to 125 μM, at different incubation times up to 72 h. The data in Figure 1 demonstrate that α-TS exerted a concentration-dependent and time-dependent cytotoxicity on leukemia lymphocytes (Figure 1A). In contrast, α-TS was not toxic towards normal lymphocytes at concentrations up to 75 μM for the entire incubation period. A slight toxicity towards normal lymphocytes was observed at 100-125 μM of α-TS (Figure 1B).
Concentration-dependent and time-dependent effect of alpha-tocopheryl succinate (α-TS) on the viability of leukemia lymphocytes Jurkat (A) and normal lymphocytes (B). The initial number of cells in all samples (before treatment) was 1×106 cells/ml. The data are the mean±SD of six independent experiments. The arrows indicate the concentration of α-TS (25 μM) that was selected for further experiments in combination with conventional anticancer drug.
A concentration of 25 μM of α-TS was selected as suitable for combination with anticancer drugs, as this concentration showed about 25-30% cytotoxicity towards leukemia lymphocytes without affecting the viability of normal lymphocytes.
Effect of α-TS on the cytotoxicity of anticancer drugs towards leukemia lymphocytes. This experiment aimed to clarify the effect of α-TS (25 μM) on the anti-proliferative activity and cytotoxicity of anticancer drugs towards the leukemia lymphocytes Jurkat. Cells were treated with α-TS and/or anticancer drugs for 24, 48 and 72 h. Cell proliferation is presented in Figure 2A, C and E. The method of data processing is described in detail in our previous article (5). To distinguish the synergistic cytotoxic effect from antagonistic/additive effects, we calculated the effect of each combination on cell proliferation as a percentage of the effect of the respective drug applied alone and compared this effect to that of α-TS applied alone. In the case of drug plus α-TS, the data located to the left of the red line on Figure 2B, D, and F represent synergistic anti-proliferative and cytotoxic effect, while the data located to the right of the red line represent antagonistic effect. All data matching the red line reflect additive effect.
Effect of alpha-tocopheryl succinate (α-TS) and anticancer drugs alone and in combination on Jurkat cell proliferation. (A, C, E) Effect of α-TS, anticancer drug and their combination on cell proliferation at different incubation times: (A) 24 h; (C) 48 h; (E) 72 h. Incubation conditions: 1×106 cells/ml, α-TS (25 μM) and/or drug (at concentrations provided in the Materials and Methods), at 37°C in a humidified atmosphere of 5%CO2. The data are the mean±SD of six independent experiments. (B, D, F) Effect of each combination (drug plus α-TS) on cell proliferation as a percentage of the effect of the drug alone, calculated at different incubation times: (B) 24 h; (D) 48 h; (F) 72 h. The dotted lines indicate the effect of α-TS on proliferation of leukemia lymphocytes as a percentage of the control (untreated cells). *In this experiment, the concentration of barasertib was 50 nM.
Most combinations (α-TS plus anticancer drug) were characterized by additive or antagonistic suppression of proliferation, as well as additive or antagonistic cytotoxicity towards Jurkat cells (Figure 2B, D, and F). Three combinations exerted a time dependent synergistic antiproliferative and cytotoxic effect: α-TS plus barasertib, bortezomib or lonafarnib. The best synergistic cytotoxicity and suppression of cell growth were observed following incubation of cells with the combination of α-TS and barasertib for 48 and 72 h (Figure 2D and F).
Effect of α-TS on barasertib-induced oxidative stress and apoptosis in leukemia and normal lymphocytes. Barasertib is a highly selective inhibitor of aurora B kinase, discovered and described in 2007 (30). Little is known regarding the molecular mechanisms of anticancer activity of barasertib, except that it provokes cell-cycle arrest and apoptosis, and increases the response to chemotherapy (30, 31). The IC50 values of barasertib on cancer cells are below 1 μM. In our previous studies, we established that barasertib in combination with other redox-modulators induced apoptosis in leukemia lymphocytes via ROS-independent (in case of 2-deoxy-D-glucose, 6-aminonicotinamidе, melatonin, and menadione/ascorbate) or ROS-dependent mechanisms (in case of docosahexaenoic acid and resveratrol) (5-9).
There is no evidence about the effect of α-TS on barasertib-induced cytotoxicity in cancer cells, as well as the effect of the combination on the viability of normal cells. In our study, treatment of normal and leukemia lymphocytes with 10 nM barasertib had no effect on the viability of either cell type (Figure 3A and C). However, treatment of leukemia lymphocytes with barasertib plus α-TS for 48 h resulted in strong cytotoxicity and impressive induction of apoptosis, accompanied by an increased production of ROS, but insignificant changes in protein-carbonyl levels (Figure 2A and B). This combination did not alter the viability and did not induce oxidative stress and apoptosis in normal lymphocytes (Figure 3C and D).
Effects of alpha-tocopheryl succinate (α-TS, 25 μM), barasertib (10 nM) and their combination on cell viability, levels of reactive oxygen species (ROS), levels of protein-carbonyl products (PC products), and apoptosis in leukemia lymphocytes (A, B) and normal lymphocytes (C, D), measured after 24 and 48 h of incubation at 37°C in humidified atmosphere of 5% CO2. The data are the mean±SD from three independent experiments. The dotted arrows indicate the level of each parameter in the control (untreated) cells, which is considered 100%.
Discussion
In this study we confirmed that α-TS inhibits proliferation and induces apoptosis and cytotoxicity in leukemia lymphocytes, without affecting of viability of normal lymphocytes. Based on our knowledge, this study is the first to report a strong synergistic cytotoxic effect towards leukemia lymphocytes with the combination of α-TS and barasertib – a selective inhibitor of aurora B kinase, bortezomib – proteasome inhibitor, or lonafarnib – farnesyltransferase inhibitor. No harmful side effects, such as induction of oxidative stress and pronounced apoptosis, were observed following treatment of normal lymphocytes with α-TS plus barasertib. These data suggest that α-TS could be a promising supplement in second line anticancer therapy, especially in acute lymphoblastic leukemia, for reducing the therapeutic doses of barasertib, bortezomib and lonafarnib and minimizing their side effects. Our results also suggest that α-TS is an appropriate carrier (matrix) for the development of new generation drug delivery systems for cancer therapy, and that it itself may have an anticancer effect.
Although the molecular target(s) of α-TS have not been identified, its selective cytotoxicity towards cancer cells has been described in many studies (10, 15-21). For example, Kumar et al. have reported selective cytotoxicity of α-TS, administered at concentrations of 25 and 50 μM to freshly isolated leukemia cells obtained from patients with chronic myeloid leukemia and peripheral blood mononuclear cells derived from normal healthy blood donors (32). Similar data have been reported by Ruiz-Moreno et al. (27). The authors have investigated a water-soluble derivative of vitamin E, D-α-tocopheryl polyethylene glycol 1000 succinate, which is commonly used as a drug delivery vehicle. In their study, human peripheral blood lymphocytes and Jurkat leukemia lymphocytes were treated with different concentrations of this vitamin E analogue (from 10 to 80 μM) and dose-dependent apoptotic cell death was observed in leukemia lymphocytes but not in normal lymphocytes. Another study evaluated the effect of α-TS on cultured human promyelocytic leukemia cells (HL-60) and induction of apoptosis by modulating mitochondrial membrane function and activating a caspase cascade was observed (33).
There are different hypotheses regarding the selectivity of α-TS for cancer cells. One of them is related to the unique chemical structure and physicochemical properties of this tocopherol ester. It is well known that the α-TS is a weak acid with a low pKa value. In a neutral pH medium, as in normal cells, α-TS usually exists in a charged, deprotonated state, which makes it difficult to pass through the plasma membrane. The acidic pH of cancer cells may favor the protonation of α-TS, which will promote its free diffusion into cells (10). This hypothesis is supported by a study demonstrating that lowering the pH of the cell culture medium increases α-TS-induced cancer cell mortality, but this does not apply to γ-tocotrienol (an apoptogenic vitamin E analogue), because it cannot to be deprotonated (34). According to other authors, the selective cytotoxicity of α-TS towards cancer cells is related to its existence in these cells in the original non-hydrolytic form, while normal cells hydrolyze α-TS, turning it into α-tocopherol, which has pronounced antioxidant properties and is non-apoptogenic (25).
The most widely discussed mechanism for the anti-proliferative activity and cytotoxicity of α-TS towards cancer cells is related to the influence of the atypical metabolism of cancerous mitochondria, which is characterized by succinate accumulation, altered redox state of complexes I and III, and reversal of the Krebs cycle (35). It has been shown that α-TS could affect the function of complex II of the mitochondrial respiratory chain by displacing ubiquinone from its binding site between the transmembrane subunits of succinate dehydrogenase (25). Thus, α-TS can impair mitochondrial function. In addition, it has been reported that the cytotoxicity of α-TS towards cancer cells can be abolished by mitoquinone (MitoQ) – a modified ubiquinone that interacts with ubiquinone binding sites of complex II (36). MitoQ has low substrate affinity and activity against complexes I and III, but it is a highly efficient redox substrate of complex II (36). Thus, complex II of the mitochondrial electron transport chain may be a possible target for α-TS. The interaction between α-TS and complex II can lead to altered intracellular redox homeostasis, mitochondrial respiration, and ATP production, and subsequent inhibition of cell proliferation. A commonly used practice in the treatment of cancer is to combine several anticancer agents with different molecular targets and mechanisms. This approach allows: (i) minimizing the induction of apoptosis, ferroptosis or necrosis in normal cells; (ii) overcoming resistance to therapeutic agents; (iii) decreasing the therapeutic doses of individual drugs, which would minimize and even eliminate the potential side effects of treatment (10, 14). However, it is still difficult to avoid harmful side effects on normal cells and tissues. In this context, the administration of natural products with redox-modulating properties can cause specific and selective changes in the redox homeostasis of cancer cells only, increase their sensitivity to conventional drugs, and even induce a synergistic cytotoxicity (5-9). This therapeutic strategy has a potential to improve the efficacy of therapy, reduce the harmful side effects, and improve the quality of life of cancer patients.
Aurora B kinase belongs to serine/threonine protein kinases, which plays a key role in chromosome segregation and synergistically regulates survival and proliferation of leukemia and lymphoma cells by regulating the following signaling pathways: AKT, mTOR and Notch (37). Aurora B kinase is thought to initiate cancer progression by indirectly inhibiting p53 activity and antagonizing apoptosis by reducing the expression of the protein cyclin dependent kinase inhibitor p21 (CIP1/WAF1) (30, 31, 38, 39). Both transcription factors are tightly connected to the mitochondrial metabolism and functionality. It should be noted that α-TS has been found to induce apoptosis in some types of cancer cells via activation of p53 (39). In other types of cancer cells, α-TS induces apoptosis even of p53(–/–) and p21(–/–) cell mutants (40). Our study indicates that these effects can be amplified when cancer cells are treated with the combination of barasertib with α-TS, which may explain the synergistic antiproliferative activity and cytotoxicity in leukemia lymphocytes, that is accompanied by impressive induction of apoptosis (Figure 3B).
Acknowledgements
This study was partially supported by the following projects: Japanese Society for the Promotion of Science (JSPS) (Grand-in-aid “Kakenhi-C”, granted to R.B.), Japan Agency for Medical Research and Development (AMED) (Project for Cancer Research and Therapeutic Evolution, P-CREATE, no. 16 cm0106202h0001), and Trakia University, Bulgaria (Grant No. FVM 06/2021).
Footnotes
Authors’ Contributions
ZZ and RB conceived the idea for the study. DI and RB wrote the first draft of manuscript. DI, DL, GZ and RP conducted the experiments. IA was involved in the critical review of the drafts and final version of the article. All Authors read and approved the final version of the manuscript
Conflicts of Interest
No potential conflicts of interest are disclosed regarding this study.
- Received October 6, 2021.
- Revision received October 24, 2021.
- Accepted October 25, 2021.
- Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
