Abstract
Background/Aim: We describe a pharmacological strategy for selectively targeting glioblastoma using a redox-active combination drug menadione/ascorbate (M/A), compared to the chemotherapeutic standard-of-care temozolomide (TMZ). Materials and Methods: Experiments were conducted on glioblastoma mice (GS9L cell transplants – intracranial model), treated with M/A or TMZ. Tumor growth was monitored by magnetic resonance imaging. Effects of M/A and TMZ on cell viability and overproduction of mitochondrial superoxide were also evaluated on isolated glioblastoma cells (GS9L) and normal microglial cells (EOC2). Results: M/A treatment suppressed tumor growth and increased survival without adverse drug-related side effects that were characteristic of TMZ. Survival was comparable with that of TMZ at the doses we have tested so far, although the effect of M/A on tumor growth was less pronounced than that of TMZ. M/A induced highly specific cytotoxicity accompanied by dose-dependent overproduction of mitochondrial superoxide in glioblastoma cells, but not in normal microglial cells. Conclusion: M/A differentiates glioblastoma cells from normal microglial cells, causing redox alterations and oxidative stress only in the tumor. This easier-to-tolerate treatment has a potential to support the surgery and conventional therapy of glioblastoma.
The treatment of brain tumors is a huge challenge for the clinic and pharmacy. New therapeutic strategies are being developed, but so far success has been unsatisfactory. Glioblastoma is the most common type of primary brain tumors, malignant and highly aggressive, with a very poor prognosis. The median survival after diagnosis is about 5 months for untreated patients and does not exceed 18-22 months after treatment (1, 2). Only 25% of patients with glioblastoma survive more than one year, and only 5% of patients survive more than five years (3).
Currently, the worldwide acclaimed “gold standard” for the first line treatment of patients with glioblastoma is surgical resection combined with radiotherapy and chemotherapy with temozolomide (TMZ) (4, 5). TMZ is a DNA alkylating anticancer drug with excellent ability to penetrate the blood-brain barrier, known to induce cell cycle arrest and apoptosis (6). Although the inhibitory effect of TMZ on tumor growth is impressive, the systemic side effects are severe: vomiting, nausea, fatigue, headache, hair loss, cachexia, and others (7). Like all DNA alkylating agents, the specificity of TMZ is relatively low and cytotoxicity affects all dividing normal cells in the organism such as immune, epithelial, and normal stem cells (8-11). This poses a risk of developing immune deficiency and serious comorbidities from this anticancer therapy. For example, TMZ is known to induce depletion of T-lymphocytes and natural killer cells and subsequent lymphopenia and myelosuppression in patients with malignant glioma (8-11). The drug also induces anemia (10, 11). In addition, TMZ is cytotoxic to non-proliferating cells such as normal astrocytes, although the effect is less pronounced than in glioblastoma cells (12, 13). The new modified derivatives of TMZ show a better therapeutic effect, but still modest in terms of the median survival and improving the quality of life of the patients with glioblastoma (14). At present, the genesis, development and progression of glioblastoma and its resistance to standard therapy remain unclear.
Redox imbalance has been found in glioblastoma. The tumor is characterized by high oxidative capacity due to their elevated glycolytic and mitochondrial oxidative metabolism (15-17). The studies suggest that redox environment plays an important role in the initiation, progression, and regression of glioblastoma, and new alternative redox therapies should be the focus of scientists, pharmacists, and clinicians. New therapeutic strategies are designed to modulate redox signaling in these tumors and potentiate the effect of standard therapy (15, 18-20).
Most of the redox-active compounds found to be cytotoxic to glioblastoma cells are characterized by the following effects: overproduction of reactive oxygen species (ROS), including overproduction of mitochondrial ROS, decrease in mitochondrial potential, and/or depletion of glutathione (15). However, most of them have not been studied for selectivity towards glioblastoma cells only and off-target effects limit their prospects.
In this article we describe a highly specific targeting of glioblastoma, using the redox-active combination drug menadione/ascorbate (M/A, 1/100 mol/mol ratio). Many studies have shown that in pharmacologically achievable doses, this combination is harmless to normal cells and tissues and well tolerated by experimental animals (21-24). Our experiments were conducted in vivo on GS9L intracranial xenograft model in mice and in vitro on glioblastoma cells and normal microglial cells. Based on our knowledge, this is the first article demonstrating the redox targeting of glioblastoma in vivo by the quinone/ascorbate combination (in particular, M/A), comparing its effect on tumor growth and animal survival with that of TMZ – a drug that, after more than 20 years, is still considered the clinical standard-of-care.
Materials and Methods
Chemicals. L-Ascorbic acid, menadione, and temozolomide were purchased from Sigma-Aldrich (Weinheim, Germany). All reagents, used in the experiments, were “analytical grade” or “HPLC-grade”.
Cells and treatment protocol. The experiments were performed on normal glial cells (EOC2) purchased from the American Type Cultured Collection (ATCC®, San Diego, CA, USA) and glioblastoma cells (GS9L) purchased from the European Collection of Authenticated Cell Cultures (ECACC General Cell Collection, Salisbury, UK). EOC2 cells were cultured in DMEM (Sigma-Aldrich), supplemented with 10% FBS (Sigma-Aldrich). GS9L cells were cultured in DMEM, supplemented with 10% FBS and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin) (Sigma-Aldrich). The concentration of glucose in the medium was standard (2 mM). All cells were cultured in a humidified atmosphere at 37°C, saturated with 5% CO2.
To remove the cells from the plates, we used a trypsin-EDTA solution (0.05% of trypsin/EDTA) (Sigma-Aldrich) and subsequent washing with phosphate-buffered saline (PBS, pH 7.4). The cells were collected by centrifugation (800 × g for 5 min) and placed in a fresh medium without antibiotics prior to treatment with the respective substance. The cells (3×105 cells/ml) were incubated with drug for different time intervals in a cell incubator. At each time interval, aliquots were used for analyses.
Ascorbate was dissolved in PBS (10 mM, pH 7.4). Menadione and temozolomide were dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich) to 10 mM stock solutions and then several working solutions in PBS were prepared. The final concentration of DMSO in the cell suspensions was below 1%. At this concentration, DMSO did not affect cell viability.
Cell proliferation and viability assays. Cell proliferation and viability was analyzed using CellTiter-Glo™ luminescent cell viability assay (Promega, Madison, MI, USA). Briefly, 100 μl aliquots of cell suspensions were placed in 96-well plates and incubated with the drug for 24, 48, and 72 h, in a humidified atmosphere (at 37°C, 5% CO2). One hundred μl of CellTiter-Glo™ reagent (containing luciferin and luciferase) were added to each well, followed by incubation using the protocol recommended by the manufacturer. The luminescence, produced by the luciferase-catalyzed conversion of luciferin into oxyluciferin by living cells, was detected using a microplate reader (TECAN Infinite® M1000, Vienna, Austria), working in a chemiluminescent mode. The linear range for this assay was up to 5×105 cells per well.
Mitochondrial superoxide assay. MitoSOX™ Red Mitochondrial Superoxide Indicator (Molecular Probes, Invitrogen, Eugene, OR, USA) is a fluorogenic probe for highly selective detection of superoxide in the mitochondria of live cells. The probe is a dihydroethidium derivative, containing triphenylphosphonium group. Once in the mitochondria, MitoSOX™ Red reagent is oxidized by superoxide and exhibits red fluorescence. The probe is not oxidized by other reactive oxygen and nitrogen species (ROS/RNS), and its oxidation is prevented by superoxide dismutase (25).
Briefly, MitoSOX™ Red was dissolved in DMSO to 5 mM stock solution, which was diluted with Hank’s Balanced Salt Solution (HBSS, containing Ca2+ and Mg2+) to prepare 3 μM MitoSOX™ Red working solution on the day of experiment. One ml of cells (5×105 cells/ml) was collected by centrifugation and the pellet was re-suspended in 1 ml of 3 μM of MitoSOX™ Red. The samples were incubated for 30 min at room temperature, protected from light, washed three times with PBS using centrifugation, and finally re-suspended in 1 ml of PBS. The fluorescence intensity was detected immediately at λex=510 nm and λem=580 nm, using a microplate reader (TECAN Infinite® M1000).
Animals and treatment protocol. The animal experiments in this study were approved by the National Institute of Radiological Sciences Institutional Animal Care and Use Committee, Chiba, Japan, and all experiments were performed in accordance with relevant guidelines and regulations. BALB/c nude mice were obtained from Charles River Labs (Japan). All mice were male and were used at 6-8 weeks of age and maintained in specific pathogen-free conditions.
The experimental design is shown in Figure 1. Glioblastoma GS9L cells (5×105 in 10 μl per mouse) were inoculated into the brain of anesthetized mice using automated injector (KD Scientific, Holliston, MA, USA) – 2 mm to the right and 1 mm anterior from the bregma, at depth of 3 mm within 10 min. The glioblastoma mice were assigned to the following groups: (i) control group – single intracranial injection of saline solution; (ii) 1× M/A-treated group – single intracranial injection of 14 μg/1.4 mg of M/A per kg body weight; (iii) 5× M/A-treated group – single intracranial injection of 70 μg/7 mg of M/A per kg body weight; (iv) TMZ-treated group – single intracranial injection of 4 mg per kg body weight. The volume of all intracranial injections was 10 μl. The mice in M/A-treated groups were also subjected to oral administration of M/A in the drinking water (150 mg/15 g of M/A per 1 liter; everyday freshly prepared except on weekends). The mice in the Control group and TMZ-treated group received M/A-free water. One day before cell transplantation, the mice were placed on vitamin C and menadione deficient diet (CLEA Inc., Tokyo, Japan).
Schematic representation of the experimental design: Treatment of mice with menadione/ascorbate (M/A), temozolomide (TMZ), or saline solution (Control).
Before injection of the drug, the tumor was visualized in the brain of each mouse using T2-weighted (T2W) MRI and the initial tumor size was calculated. Body weight was measured once or twice per week. MRI measurements were performed once per week. The approved humane endpoint was two months after cell transplantation. However, the mice were sacrificed at the following conditions: when the tumor size exceeded 100 mm3, at rapid weight loss of 25%, headedness and/or tetraplegia.
In vivo MRI measurements. Each mouse was anesthetized with isoflurane (3% for initial induction and 1-2% during MRI scanning) and placed in the prone position on a custom-built MRI stage with a bite bar and a facemask. The respiration rate was monitored using a respiration sensor (SA Instruments, Inc., NY, USA) and regulated at 80-120 breaths per minute. The core body temperature was monitored with a rectal probe (FOT-M and FTI-10, FISO Technologies Inc., Germany) and regulated at 37.0±1.0°C using a water-circulating pad and a warm circulation air system. MRI data were acquired using a horizontal 7.0-T Bruker BioSpec 70/40 MRI system with an 86 mm volume transmit and a 4-channel phased array receive-only cryoprobe (Bruker Biospin, Ettlingen, Germany). The software and console of the MRI scanner was ParaVision 360 and AVANCE NEO, respectively. Following the standard adjustment routines, pilot scans (Tripilot sequence) were used for accurate positioning of the animal head inside the magnet.
The T2W images were obtained using a spin-echo 2D-RARE (rapid acquisition with relaxation enhancement) pulse sequence with the following parameters: repetition time=3,000 ms, effective echo time=60 ms, RARE factor=8, field of view=16×16 mm2, matrix size=160×160, in-plane resolution=0.1×0.1 mm2, number of slices=13, slice thickness=0.3 mm, slice gap=0 mm, fat suppression=on, and number of averages=8.
Statistical analysis. All results are expressed as means±standard deviation (SD). The normality of the distribution for all parameters of each experimental group in vivo was initially confirmed by using Kolmogorov-Smirnov test. The most extreme differences for all experimental groups were below the critical D-values. Based on the normality of distribution in all groups, the comparisons between them were performed using Student’s t-test for multiple comparisons. Two-tailed p-values of less than 0.05 were considered statistically significant.
Results
Mitochondria-mediated anticancer effect of M/A and TMZ – cancer cells versus normal cells. Glioblastoma cells and normal microglial cells were treated with different concentrations of M/A or TMZ and cell proliferation and/or viability were analyzed within 24-72 h (Figure 2). In GS9L cells, M/A exhibited a strong cytotoxic effect at high concentrations (≥5/500 μM/μM) and a cytostatic effect at low/tolerable concentrations (<5/500 μM/μM) (Figure 2A). In EOC2 cells, M/A did not exhibit a cytotoxic effect up to 20/2000 μM/μM (Figure 2B). IC50 values were over 20/2000 and ~8.7/870 μM/μM of M/A for EOC2 and GS9L cells, respectively. The comparative analysis shows that M/A exhibits selective cytotoxicity towards glioblastoma cells without significant effect on the viability of normal microglial cells.
Concentration-dependent and time-dependent effects of menadione/ascorbate (M/A) and temozolomide (TMZ) on cell proliferation and viability, as well as the production of mitochondrial superoxide in glioblastoma and normal microglial cells: (A) proliferation/viability of GS9L cells treated with M/A; (B) viability of EOC2 cells treated with M/A; (C) level of mitochondrial superoxide in M/A-treated GS9L and EOC2 cells; (D) proliferation/viability of GS9L cells treated with TMZ; (E) viability of EOC2 cells treated with TMZ; (F) level of mitochondrial superoxide in TMZ-treated GS9L and EOC2 cells. The values of all parameters in untreated (control) samples were considered as 100%. Data are means±SD from three independent experiments with two parallel measurements for each experiment.
In GS9L cells, M/A induced dose-dependent overproduction of mitochondrial superoxide and severe oxidative stress, especially at concentrations over 5/500 μM/μM (Figure 2C, red columns). In EOC2 cells, M/A induced a relatively low and dose-independent increase in mitochondrial superoxide and mild oxidative stress, which seemed to be well tolerated (Figure 2C, blue columns).
In contrast, TMZ exhibited cytotoxic effect in both glioblastoma and normal microglial cells at ≥100 μM concentration (Figure 2D and E). The cytotoxicity of TMZ on normal microglial cells was less pronounced, but the IC50 values were comparable (~85 μM versus ~75 μM of TMZ for EOC2 versus GS9L cells, respectively). TMZ induced impressive overproduction of mitochondrial superoxide in glioblastoma cells (~20 time over the control level at 100 μM of TMZ), but the same value was also detected in normal microglial cells (Figure 2F). Obviously, the drug can not distinguish glioblastoma cells from normal microglial cells and causes severe oxidative stress in both cell lines.
Effect of M/A and TMZ on tumor growth and survival in mice with glioblastoma. The mice were inoculated with GS9L glioblastoma cells and after 4 days, tumors were visualized in all animals, using MRI. The initial tumor size was 0.75±0.13 mm3 (early stage of tumor). The mice were divided into three groups: M/A-treated group – single intracranial injection of M/A plus daily oral administration of M/A in drinking water; TMZ-treated group – single intracranial injection of TMZ; Control group – single intracranial injection of saline solution. M/A-treated group was divided in two subgroups: (i) treated with low dose of M/A (1×, equal to 14 μg/1.4 mg per kg body weight); and (ii) treated with high dose of M/A (5×, equal to 70 μg/7 mg per kg body weight).
The tumors were visualized, and their size was measured each week after cell transplantation, using T2W MRI (Figure 3). In the control group, the exponential tumor growth was observed after 10-14 days, and the humane endpoint (>100-120 mm3) was reached after 17-20 days. In 1× M/A-treated group, we observed the same tendency as in the control group, but the tumors reached >100 mm3 after 28-30 days. In the 5× M/A-treated group, the tumors grew slowly during the first 3 weeks of cell transplantation. The exponential growth was observed after that, and reached a size of ~100 mm3 after 33-45 days.
Effect of menadione/ascorbate (M/A) and temozolomide (TMZ) on tumor growth in mice with early-stage glioblastoma. (A) Effect of single intracranial injection of M/A and TMZ on tumor growth in the brain of GS9L glioblastoma-grafted mice, detected by magnetic resonance imaging (MRI) within 28 days after cell transplantation and 24 days after drug administration. Control mice were injected intracranially with the same volume of saline solution. 1× M/A – 14 μg/1.4 mg per kg body weight; 5× M/A – 70 μg/7 mg per kg body weight; TMZ – 4 mg per kg body weight. The drugs were administered on day 4th of the brain cell transplant, when the tumor size was about 0.75±0.13 mm3. Number of mice in each experimental group: Control group (n=5); 1× M/A-treated group (n=3); 5× M/A-treated group (n=4); TMZ-treated group (n=5). Data are means±SD from 3 mice at each time-point. #In the control group, the last measurement of tumor size by MRI was on day 15th or 17th, depending on their health condition and endpoint. All values in the groups treated with M/A or TMZ were statistically significant (p<0.05) compared to the control group on the first week and beyond. (B) Effect of single intracranial injection of M/A and TMZ on tumor growth in the brain of GS9L glioblastoma-grafted mice, detected by MRI before and 3 days after drug administration at the same conditions described in (A). All values on day 3rd were statistically significant versus the values on day 0 (p<0.05 for M/A-treated groups; p<0.01 for TMZ-treated group). (C) Representative T2-weighted magnetic resonance images of the brain of control mouse and M/A-treated mouse.
Representative images of the brain of control and 5× M/A-treated mice, obtained after 17 and 21 days of cell transplantation, respectively, are shown in Figure 3C. The tumor size in the control mouse was ~86 mm3, while that in the M/A-treated mouse was ~15 mm3. The survival of the same control mouse was 18 days versus 35 days for M/A-treated mouse.
In TMZ-treated group, the tumor grew very slowly and reached a maximum recorded size of 8.4±0.8 mm3 in the fourth week of cell transplantation (Figure 3A). Three days after intracranial injection of M/A or TMZ, the size of the tumors decreased significantly compared to the initial size (Figure 3B). For this short time after the injection, the two drugs reduced the size of the tumor equally. However, the overall effect of M/A on tumor growth was less pronounced than that of TMZ.
Surprisingly, the effect of M/A on animal survival was comparable to that of TMZ, and even better in some mice (Figure 4A). In both drug-treated groups, the median survival was significantly higher compared to controls: 33.7±6.0 days for M/A and 32.3±0.6 days for TMZ versus 18.8±0.8 days for controls (Figure 4B).
Effect of menadione/ascorbate (M/A) and temozolomide (TMZ) on survival and body weight of mice with glioblastoma. (A) Effect of single intracranial injection of M/A and TMZ on survival of GS9L glioblastoma-grafted mice. Data are means±SD from: 5 mice in the Control group; 7 mice in the M/A-treated group; and 5 mice in the TMZ-treated group. (B) Median survival of mice in the groups described in (A): ***p<0.001 versus control group. (C) Dynamics of body weight in control, M/A-treated, and TMZ-treated GS9L glioblastoma-grafted mice. Red arrows indicate the time of injection (day 4th after cell transplantation). At each time-point, the data are means±SD from: 3-5 mice in the Control group; 4-7 mice in the M/A-treated group; and 5 mice in the TMZ-treated group, depending on their survival. M/A-treated group combines 3 mice treated with 1× M/A and 4 mice treated with 5× M/A. #In the control group, the last measurement of body weight was within days 15th-20th, depending on their health condition and endpoint.
Mice in the control group lost weight after the 7th day of cell transplantation, reaching ~16.0 g at the endpoint (Figure 4C). Mice in the TMZ-treated group lost weight drastically after drug administration, reaching ~13.0 g at the endpoint. M/A-treated mice lost weight immediately after the intracranial injection and regained weight thereafter.
It should be noted that we observed severe adverse side-effects in TMZ-treated mice soon after drug administration, such as: fever (37.5-38.0°C), dizziness, convulsions, and tetraplegia. No such side-effects were detected in M/A-treated mice. Harmful side-effects and rapid weight loss in TMZ-treated mice are the most likely reason of their slightly shorter survival (~32 days) at small tumor size, compared to the mice treated with 5× M/A (~37 days) but with bigger tumor size.
In a pilot study, we compared the effect of single intracranial injection of TMZ and 5× M/A on tumor growth in mice, when the treatment starts at an initial tumor size of ~3.0 mm3 (moderate stage of tumor) – day 9th of cell transplantation (Figure 5A). Five days after drug administration, tumors were visualized by MRI. In TMZ-treated mice, the mean tumor size was ~4.5 mm3, while in M/A-treated mice it was ~9.0 mm3. However, the survival was almost the same in both groups, which was preceded by rapid weight lost and side-effects in the TMZ-treated group, but not in the M/A-treated (Figure 5B).
Effect of menadione/ascorbate (M/A) and temozolomide (TMZ) on tumor growth and drug-related side-effects in mice with moderate-stage glioblastoma (A) Comparison between the effect of single intracranial injection of 5× menadione/ascorbate M/A (70 μg/7 mg per kg body weight) and temozolomide (TMZ) (4 mg/kg body weight) on tumor growth in the brain of GS9L glioblastoma-grafted mice, detected by magnetic resonance imaging within two weeks after cell transplantation and five days after drug administration. The drugs were administered on day 9th of the brain cell transplant, when the tumor size was ~3.0 mm3 (red arrows). Data are means±SD from two mice in each group. (B) The data in the table correspond to the mice described in (A).
Discussion
The described experimental data show that: (i) M/A induces highly selective (targeted) cytotoxicity in isolated glioblastoma cells, but not in normal microglial cells. This is accompanied by dose-dependent overproduction of mitochondrial superoxide in glioblastoma cells only. Conversely, TMZ is cytotoxic and causes severe oxidative stress in both glioblastoma and normal microglial cells.
A single intracranial injection of M/A (70 μg/7 mg per kg body weight) and subsequent oral administration in the drinking water (15 g/150 mg per liter) suppresses tumor growth. M/A increases survival without severe side effects in the mice that is characteristic for TMZ at the dose used (4 mg/kg body weight single intracranial injection). The effect of M/A on tumor growth is less pronounced than that of TMZ, but the effect of M/A on survival was comparable, and in some mice even better than that of TMZ at the dose we have tested so far.
These observations suggest that M/A may differentiate cancer cells and tissues from normal ones. Thus, the drug causes oxidative stress only in the tumor. We also found that M/A treatment (even by intravenous injection of 140 μg/14 mg per kg body weight) does not induce erythropenia and leukopenia (the data will be published elsewhere). The harmless and even beneficial effects of M/A on non-cancerous cells and tissues may be one of the reasons for better tolerance to the drug and the absence of drug-related side effects during treatment.
We found only one published study, reporting that menadione alone and in combination with ascorbate significantly suppresses DNA replication and inhibits the growth of patient-derived glioma cells (hGCL and DBTRG.05MG) with IC50 values in the range of 10-25 μM (26). One potentially critical observation is that M/A treatment completely inhibits the re-growth of glioma cells at long-term treatment in vitro, while menadione-treated cells resume their proliferation. Ascorbate administered alone in tolerable doses (≤2.5 mM) did not affect the growth of human glioma cells. However, the authors did not investigate the effects of M/A on glioma models in vivo.
Two other studies have shown the effect of high doses of ascorbate on tumor growth in glioblastoma mice. Ryszawy et al. (2019) have reported that high doses of ascorbate (≥5 mM) significantly decrease the proliferation and mobility of glioblastoma cells (U87 and T98G) (27). This effect is accompanied by overproduction of intracellular ROS and necrotic cell death. The authors have found that intravenous administration of ascorbate to glioblastoma-bearing rats inhibits tumor growth and invasion, as analyzed ex vivo. However, the injected doses of ascorbate are relatively high – 1 g and 2 g per kg body weight, which resulted in 5-10 mM of ascorbate in the body fluids. No indications of systemic side effects were reported in this study, but at these doses of ascorbate the risk of adverse effects is potentially high, according to the literature (28-30). Grasso et al. (2014) have demonstrated that a 1-hour incubation of mouse glioma cells (GL261) with 5 mM ascorbate sensitizes the cells to 6 Gy radiation, as assessed by their survival and colony formation in vitro (31). However, daily intraperitoneal injection of ascorbate (1 mg per kg body weight) with a single 4.5 Gy dose in glioma-bearing mice (GL261 intracranial model) has no effect on tumor growth as analyzed histologically ex vivo, nor does it improve survival. Rather, tumor growth is faster, and survival is shorter in glioma-bearing mice treated with radiation plus ascorbate versus those treated with radiation alone. Histological analysis from this study shows less necrosis in tumors treated with both radiation and ascorbate, suggesting a radio-protective effect of pharmacological ascorbate in vivo. The authors explain the discrepancies between their in vitro and in vivo data by differences in the tumor microenvironment, which determines whether ascorbate remains outside the cells acting as a prooxidant, or it enters the cells and acts as an antioxidant.
Our recent studies suggest that ascorbate should not be considered simply as a prooxidant or antioxidant, as well as menadione should not be considered simply as a coagulant (32, 33). Ascorbate is one of the most abundant cytosolic redox-active compounds and could serve as a “buffer” of excess-reducing equivalents in the intracellular aqueous phase of cancer cells due to their oxidative environment. Ascorbate could also be a modulator of mitochondrial respiration acting as an “injector” of electrons, mainly for cytochromes (c and b) and especially in the presence of selected quinones (such as menadione), inducing reverse electron transport in altered and overcharged cancerous mitochondria (21, 33). The doses of menadione suppressing tumor growth are significantly lower than those used as a coagulant.
Menadione and ascorbate are powerful redox cyclers and administered alone or in combination, they can induce intracellular production of ROS by interaction with molecular oxygen (21, 34, 35). It is generally accepted that the M/A combination causes cancer cell death by induction of oxidative stress and subsequent replicative stress (36-38). This mechanism has also been described in M/A-treated glioma cells in vitro (26).
Recently, we found that M/A exhibits a highly specific and synergistic suppression on cancer cell growth and viability, without adversely affecting the viability of normal cells at pharmacologically achievable concentrations (21). These experiments were performed on several cancer and normal cells lines of origin rather than glioblastoma and normal microglial cell lines. In this case, the targeted anticancer effect of M/A was the result of a selective redox cycling between both substances within dysfunctional mitochondria, which was accompanied by induction of severe oxidative stress in cancer cells only (21, 39). The M/A-induced cytotoxicity in cancer cells was also accompanied by: (i) an extremely high production of mitochondrial superoxide; (ii) a significant decrease of mitochondrial membrane potential and steady-state levels of ATP; (iii) a significant depletion of succinate, NADH, NAD+, and glutathione. All these effects were dose-dependent and irreversible at pharmacological doses of M/A (21).
We suppose that targeted anticancer effects of M/A are strongly influenced by the conversion of menadione to menaquinone (vitamin K2) via UBIAD1-catalyzed prenylation in normal cells, but not in cancer cells (40, 41). Down-regulation of UbiA prenyltransferase domain containing protein 1 (UBIAD1), also known as transitional epithelial response protein 1 (TERE1), is a hallmark of multiple cancers, according to The Human Protein Atlas (UBIAD1 protein expression summary - The Human Protein Atlas; https://www.proteinatlas.org/ENSG00000120942-UBIAD1). Based on these data, we assume that the targeted anticancer effect of M/A is due to their specific redox cycling in the dysfunctional mitochondria of cancer cells only, which is accompanied by a severe oxidative stress and an impairment of their “pro-oncogenic” functionality. This mechanism appears to be valid for glioblastoma. However, it should be noted that there is evidence to suggest that the mechanism of targeting glioblastoma by M/A is complex and likely not limited simply to modulating tissue redox state and overproduction of ROS by menadione and/or ascorbate in cancer cells.
Acknowledgements
The study was partially supported by the following grants: QST/ICM grant funded by IC-MedTech Corp., US (granted to R.B.); AMED Grant Number 16cm0106202h funded by the Japanese Agency for Medical Research and Development; and Kakenhi (#21H04966, 17KK0102) funded by the Japanese Agency for Promotion of Science. The participation of Dr. Takako Maruoka (QST, Japan) in the preparation of GS9L glioblastoma model is gratefully acknowledged.
Footnotes
Authors’ Contributions
ZZ, RB, and TM conceived the idea for the study. RB and ZZ produced the first draft of manuscript. AS, SS and RB conducted the experiments. DL and GZ conducted the statistical analysis. TM and IA were involved in the critical review of the drafts and final version. All Authors read and approved the final version of the manuscript.
Conflicts of Interest
No potential conflicts of interest are disclosed in relation to this study.
- Received October 5, 2021.
- Revision received October 27, 2021.
- Accepted October 29, 2021.
- Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.