We have previously reported the in vitro and in vivo efficacy of N,N-bis(2-chloroethyl)-2-(1-methyl-1,2,3,6-tetrahydropyridin-4-yl)propenamide (MP-MUS), a prodrug that targeted the mitochondria of glioblastoma (GBM). The mitochondrial enzyme, monoamine oxidase B (MAOB), is highly expressed in GBM and oxidizes an uncharged methyl-tetrahydropyridine (MP-) moiety into the mitochondrially targeted cationic form, methyl-pyridinium (P+-). Coupling this MAOB-sensitive group to a nitrogen mustard produced a prodrug that damaged GBM mitochondria and killed GBM cells. Unfortunately, the intrinsic reactivity of the nitrogen mustard group and low solubility of MP-MUS precluded clinical development. In our second-generation prodrug, MP-Pt(IV), we coupled the MP group to an unreactive cisplatin precursor. The enzymatic conversion of MP-Pt(IV) to P+-Pt(IV) was tested using recombinant human MAOA and rhMAOB. The generation of cisplatin from Pt(IV) by ascorbate was studied optically and using mass spectroscopy. Efficacy toward primary GBM cells and tumors was studied in vitro and in an intracranial patient-derived xenograft mice GBM model. Our studies demonstrate that MP-Pt(IV) is selectively activated by MAOB. MP-Pt(IV) is highly toxic toward GBM cells in vitro. MP-Pt(IV) toxicity against GBM is potentiated by elevating mitochondrial ascorbate and can be arrested by MAOB inhibition. In in vitro studies, sublethal MP-Pt(IV) doses elevated mitochondrial MAOB levels in surviving GBM cells. MP-Pt(IV) is a potent chemotherapeutic in intracranial patient-derived xenograft mouse models of primary GBM and potentiates both temozolomide and temozolomide–chemoradiation therapies. MP-Pt(IV) was well tolerated and is highly effective against GBM in both in vitro and in vivo models.

The prognosis for patients with glioblastoma (GBM) is often grim, with limited chemotherapeutic options after surgical resection of tumors. The current standard of care is the Stupp protocol, which involves aggressive tumor resection, whole-brain/focal radiation with temozolomide, and then temozolomide chemotherapy (1, 2). Encouraging data from phase III clinical trials with drug combinations such as lomustine/temozolomide (median survival, 48.1 on lomustine/temozolomide vs. 31.4 months on temozolomide; ref. 3) or tumor treatment fields (median survival, 25.2 months; ref. 4), or immunotherapy (5) demonstrate significant recent therapeutic advances. Ultimately, resistance to temozolomide develops and patients undergo salvage therapy (chemotherapy or chemoradiotherapy), providing minimal efficacy at best, and significant toxicity.

GBM tumors tend to be hypoxic, and GBM cells are known to express active oxygen-sensing master gene controllers, HiF-1 and HiF-2 (6, 7). GBM tumors respond to hypoxia by metabolic remodeling, involving the upregulation of nonaerobic pathways, downregulation of mitochondrial respiration, and a reduction in mitochondrial mass (8, 9). The mitochondria of cancer cells generally have elevated ascorbate levels (10). These high levels result from the importation of dehydroascorbate into the cells by Glut transporters, then into mitochondria by Glut10, the SLC2A10 gene product, and subsequent reduction to ascorbate by glutathione S-transferase omega-1 or 2. Glut10 is highly expressed in GBM, consequently elevating mitochondrial ascorbate (11). GBM can acquire resistance to temozolomide via elevated mitochondrial ascorbate (12). Unlike nuclear DNA, mitochondrial DNA lack robust repair mechanisms (13) and are thus, sensitive to DNA-damaging chemotherapeutics. Mitochondria are thus very attractive chemotherapy targets and mitochondrial enzymes, such as monoamine oxidase B (MAOB; ref. 13), are being explored for drug development (14).

MAOB is located on the inner surface of the outer mitochondrial membrane and catabolizes monoamine substrates like dopamine. MAOB activity is elevated in GBM tumors (15) and expression is associated with poor patient outcomes (16). This overexpression of MAOB in GBM appears to be linked to the Warburg shift (17) and HiF-1 activation (18–20). We showed that cultured primary GBM cells have >3-fold higher MAOB activity than normal human astrocytes (NHA), and yet have 5- to 10-fold fewer mitochondria than NHAs (13).

We have previously reported the in vitro and in vivo efficacy of N,N-bis(2-chloroethyl)-2-(1-methyl-1,2,3,6-tetrahydropyridin-4-yl)propenamide (MP-MUS), an MAOB-activated prodrug that targets GBM mitochondria (21). MAOB oxidizes the uncharged methyl-tetrahydropyridine (MP-) group into its mitochondrially targeted cationic form, methyl-pyridinium (P+-). Lipophilic cations, like P+-, cross the inner mitochondrial membrane and extensively accumulate in the mitochondrial matrix, driven by the large (∼−180 mV) mitochondrial membrane potential. At 37°C, the mitochondria/cytosol accumulation ratio of [P+-] is approximately 1,000:1 (22, 23).

MP-MUS was shown to damage GBM mitochondria and kill GBM cells in culture. Unfortunately, the intrinsic reactivity of the nitrogen mustard group and low solubility of MP-MUS precluded clinical development. Our criteria for a suitable replacement for the nitrogen mustard group thus, included (i) small size allowing the prodrug to access the MAOB substrate channel, (ii) lipophilicity to cross the blood–brain barrier, (iii) low reactivity until inside the mitochondrial matrix, and (iv) ability to damage mitochondrial matrix contents.

MP-Pt(IV), our second-generation mitochondrial-targeting prodrug incorporates all our design criteria for selective delivery and activation of an inert prodrug into the mitochondrial matrix of GBM (Fig. 1). MP-Pt(IV) utilizes the same mitochondrial-targeting MP moiety (21, 24) as MP-MUS, but replaces the nitrogen mustard group of MP-MUS with an inert, asymmetric Pt(IV)-ethylene glycol chloride complex, -Pt(IV) (25). Unreactive Pt(IV) drugs can be reduced into their highly reactive Pt(II) daughter compounds by cellular ascorbate and thiols (26, 27). In silico modeling suggested that the small and hydrophobic -Pt(IV) group (28) would allow MP-Pt(IV) to circulate, cross the blood–brain barrier, and readily access the substrate pocket of MAOB, where it would be rapidly oxidized to give P+-Pt(IV).

Figure 1.

Methyl tetrahydropyridines, like MP-Pt(IV), are selectively oxidized by MAOB into their corresponding cationic pyridinium species, P+-Pt(IV), via the dihydropyridinium, MD-Pt(IV). Lipophilic cations, like P+-Pt(IV), accumulate inside the mitochondrial matrix (1,000:1, inside/outside) due to the approximately −180 mV mitochondrial membrane potential. Within the matrix, the Pt(IV) will be reduced to cisplatin by GSH (i) or by ascorbate (ii). The reaction of cisplatin with GSH to form mono- or di-adducts could deplete mitochondrial GSH levels and cause oxidative stress. Dehydroascorbate is reconverted into ascorbate by glutathione S-transferase omega-1 & 2 (iii).

Figure 1.

Methyl tetrahydropyridines, like MP-Pt(IV), are selectively oxidized by MAOB into their corresponding cationic pyridinium species, P+-Pt(IV), via the dihydropyridinium, MD-Pt(IV). Lipophilic cations, like P+-Pt(IV), accumulate inside the mitochondrial matrix (1,000:1, inside/outside) due to the approximately −180 mV mitochondrial membrane potential. Within the matrix, the Pt(IV) will be reduced to cisplatin by GSH (i) or by ascorbate (ii). The reaction of cisplatin with GSH to form mono- or di-adducts could deplete mitochondrial GSH levels and cause oxidative stress. Dehydroascorbate is reconverted into ascorbate by glutathione S-transferase omega-1 & 2 (iii).

Close modal

We expected that P+-Pt(IV) would be rapidly converted into cisplatin in GBM mitochondria due to high ascorbate levels. Within GBM mitochondria, the regenerated cisplatin could deplete glutathione [GSH, thereby impairing detoxification of reactive oxygen species (ROS)] and induce cell death (29, 30).

The differential sensitivity of GBM toward MP-Pt(IV) emerges from three key factors: (i) GBM has much higher levels of MAOB than other tissues (15, 31), restricting the generation of mitochondrially targeted P+-Pt(IV), (ii) mitochondrial matrix P+-Pt(IV) levels will be highest in cells where (MAOB:mitochondria) ratios are highest, found in GBM (17), and (iii) high ascorbate levels in the GBM matrix drive the conversion of inert Pt(IV) into cisplatin (12).

Herein, we report the synthesis and results from in vitro and in vivo studies of MP-Pt(IV) in a primary xenograft mice GBM model. We postulated that the MAOB (32) and SLC2A10 (33) genes undergo simultaneous activation via HiF-induced changes in Sp1/Sp3 levels. To examine this hypothesis, we analyzed the overall survival (OS) outcomes of patients with GBM, with respect to MAOB and SLC2A10 mRNA levels, from the major GBM transcriptome databases, The Cancer Genome Atlas (TCGA)-GBM (RNA Seq V2 RSEM), Gravendeel, and Chinese Glioma Genome Atlas.

Accumulation of MP-Pt(IV) in the mitochondrial matrix

The Nernst equation can be used to calculate the accumulation of cations in the mitochondrial matrix, driven by the approximately −180 mV mitochondrial membrane potential (ΔΨ):

formula

At 37°C, the mitochondria/cytosol accumulation ratio of [P+-] will be approximately 1,000:1 (22, 23).

Patient survival data and statistical analysis

We selected three genes to serve as proxies of mitochondrial numbers: COX15, TFAM, and MTPAP, each of which has been shown to correlate with steady-state levels of mitochondria in normal and cancer cells (34–39). Three databases were examined to determine the transcript levels of genes and patient OS (months). Optimal high/low transcript cutoffs for the generation of Kaplan–Meier survival curves were identified from the maximum difference of ranked-means and statistical significance was calculated using the log-rank χ2 test. (28)

Synthesis of MP-Pt(IV)

MP-Pt(IV) was obtained by HBTU-mediated coupling of Pt(IV)-glycol (25) and MP-carboxylic acid (21), followed by chromatographic purification, detailed in the Supplementary Data.

Mass spectra were determined on a Thermo-Scientific LCQ FLEET spectrometer in electron impact mode. Proton (1H) NMR spectra were recorded at 600 MHz on a Bruker spectrometer using the solvent (DMSO-d6) as the internal standard. J values are given in hertz (Hz). TLCs were performed using Merck glass sheets precoated with Kieselgel 60 F254 (0.2 mm) as the adsorbent and visualized with UV light (254 and 365 nm). Column chromatography was conducted under medium pressure on silica (Kieselgel 60, 240–400 mesh). Reagents were purchased from Sigma-Aldrich and used as received.

Measurements of H2O2 generation with Amplex Red/HRP

Amplex Red (150 μmol/L) and HRP (3 U/mL) were incubated with recombinant human (rh) MAOA or rhMAOB (1 U/mL) in PBS buffer, pH 7.4, at 37°C in 96-well format (200 μL volume/well). Because the small amounts of radicals generated by the photosensitization of resorufin (40) could reduce Pt(IV) into cisplatin, which in turn quenched the fluorescence of resorufin, we measured the formation of resorufin using the absorbance of 572–672 nm in a BioTek Synergy HT Spectrophotometer at t = zero and at 40 minutes.

Fixation and permeabilization

Cells were fixed in ice-cold 4% paraformaldehyde for at least 1 hour and then washed in PBS (Thermo Fisher Scientific). MitoTracker, MitoSox Red, and H2-DCF assays were performed in nonpermeabilized cells. The activity of caspase-3 in fixed, 0.1% Triton-permeabilized cells was measured using the Molecular Probes R110-EnzChek Assay Kit (catalog no., E13184), incubating cells for 1 hour at 37°C.

Primary human GBM

Human glioma cells were harvested from patients during surgical excision and isolated within 10 minutes after removal. The primary cultures used here, GBM115 and GBM157, are highly aggressive and heterogeneous, with high MGMT expression and temozolomide IC50 value (24 hours) >1 mmol/L. Tumors were homogenized with a pipette, and cells were grown for 2 weeks in DMEM supplemented with 20% FBS, GlutaMAX-I, sodium pyruvate, and penicillin/streptomycin. Glioma cells were grown to confluence 24 hours after treatment with an identical volume of the drug (in DMSO) or DMSO alone (maximum 0.04% v/v of DMSO/medium); cells were cultured in Costar 96-well Plates (Corning). After treatment, cells were grown for 24 hours in the presence or absence of all effectors (total volume of 250 μL). GBM115 and GBM157 were used at a low passage number in all in vitro experiments.

To modulate intracellular ascorbate, we preincubated primary GBM cell cultures overnight with unsupplemented control media and with media supplemented with 5 mmol/L dehydroascorbate or 5 mmol/L ascorbate. The media were replaced after 24 hours, and cells were incubated with MP-Pt(IV) for 24 hours, then given a viability dye, fixed, and counted. Additional control experiments were run after blocking MAOB activity by preincubation with 10 μmol/L selegiline followed by incubation with 14 μmol/L MP-Pt(IV) or incubation of GBM cultures with the precursor Pt(IV)-ethylene glycol at 140 μmol/L for 24 hours.

Animal models of primary brain glioma

Study approval: tumors/resulting cultures obtained from GBM tissues had no identifiable patient information under institutional review board protocol 00014547. Animal research was conducted according to the Institutional Animal Care and Use Committee (IACUC) protocol AUP-0818-0043, approved by the IACUC of Methodist Hospital (Houston, TX). All animal care procedures conformed to the Guide for the Care and Use of Laboratory Animals (National Research Council, National Academy Press, Washington DC 1996).

Transplantation of GBM into mice

We utilized a reported method for injecting human GBM cultures into the mouse brain via the postglenoid foramen (41). Primary GBM tumors were initially grown in the flanks of tumor donor mice, then excised, chopped, and homogenized in ice-cold PBS, 1:1, using a BeadBug benchtop homogenizer. The homogenized cells were diluted with a solution of Matrigel media (4:6), kept on ice, and were injected into the postglenoid foramen of 48 female athymic nude mice over 3 hours. On day 8 after xenografting, the 42 heaviest animals were selected and randomized into seven groups of 6 animals. Therapy began on day 11, and the groups underwent six different treatment regimens (Fig. 6). MP-Pt(IV) (15 mg/kg) was administered via tail vein injection in saline; temozolomide (5 mg/kg) was given by gavage, and whole-head radiation (2 Gy) was administered from a cesium source. Control group animals received vehicle by injection or gavage and were given sham radiation. All animals that received either radiation or sham radiation were first anesthetized with isoflurane inhalation, then by subcutaneous dexmedetomidine (0.5–1.0 mg/kg). Seven animals were placed in the thermostatically ballasted radiation shield then transferred into the Cs-safe, and then rod elevated (treatment) or not (sham). Postradiation/sham, animals were administered with the anesthesia antidote, atipamezole (1 mg/kg), subcutaneously.

The animals were monitored and weighed daily during the first 4 months, and then every 3–5 days thereafter. Animals were sacrificed when they reached an ethical endpoint and their brain/tumors were preserved in formaldehyde. The study was terminated on day 300 as the nude mice begin to suffer age-dependent maladies.

The aggressive GBM phenotype analyzed in silico: MAOB, mitochondrial levels, and SLC2A10

MP-Pt(IV) was designed to preferentially kill cells that have high MAOB, low levels of mitochondria, and high Glut10. Examination of GBM patient survival in the three databases as a function of MAOB levels shows that expression of MAOB is associated with poor patient outcome (Fig. 2A, i). Survival curves of tumors with high transcript levels/ratios are the top lines (circles) and low transcript levels/ratios are the bottom lines (triangles) in each graph.

Figure 2.

Kaplan–Meier survival curves demonstrating the effect of mitochondrial MAOB and SLC2A10 mRNA transcript levels on the OS of patients with GBM. Also shown is the effect on the ratio of both MAOB and SLC2A10, with respect to a mitochondria proxy, COX15. The data used to generate these survival curves were obtained from the TCGA RNA sequencing (155 patients, median survival of 11.3 months), the Gravendeel (157 patients, median survival of 8.6 months), and CGGA (222 patients, median survival of 14.1 months) databases. Survival curves of tumors with high transcript levels/ratios are the top lines (blue circles), and low transcript levels/ratios are the bottom lines (red triangles) in each graph. The Kaplan–Meier curves were analyzed using the χ2 log-rank test, and the calculated P value is indicated on each plot and in all cases, P < 0.02. The bottom lines in each panel represent high levels (bad for patients), and top lines represent low levels (good for patients). A, High levels of MAOB mRNA transcripts are indicative of poor patient outcomes (i). Low expression of COX15 (heme a synthase) is positively correlated with survival (ii). High levels of MAOB per mitochondria, the MAOB/COX15 ratio, are indicative of poor prognosis (iii). B, The mitochondrial dehydroascorbate transporter, Glut10 (SLC2A10), is a risk factor in patients with GBM (i). High SLC2A10/COX15 ratios are also indicative of GBM aggressiveness (ii).

Figure 2.

Kaplan–Meier survival curves demonstrating the effect of mitochondrial MAOB and SLC2A10 mRNA transcript levels on the OS of patients with GBM. Also shown is the effect on the ratio of both MAOB and SLC2A10, with respect to a mitochondria proxy, COX15. The data used to generate these survival curves were obtained from the TCGA RNA sequencing (155 patients, median survival of 11.3 months), the Gravendeel (157 patients, median survival of 8.6 months), and CGGA (222 patients, median survival of 14.1 months) databases. Survival curves of tumors with high transcript levels/ratios are the top lines (blue circles), and low transcript levels/ratios are the bottom lines (red triangles) in each graph. The Kaplan–Meier curves were analyzed using the χ2 log-rank test, and the calculated P value is indicated on each plot and in all cases, P < 0.02. The bottom lines in each panel represent high levels (bad for patients), and top lines represent low levels (good for patients). A, High levels of MAOB mRNA transcripts are indicative of poor patient outcomes (i). Low expression of COX15 (heme a synthase) is positively correlated with survival (ii). High levels of MAOB per mitochondria, the MAOB/COX15 ratio, are indicative of poor prognosis (iii). B, The mitochondrial dehydroascorbate transporter, Glut10 (SLC2A10), is a risk factor in patients with GBM (i). High SLC2A10/COX15 ratios are also indicative of GBM aggressiveness (ii).

Close modal

Mitochondrial proxy transcripts for COX15, TFAM, and MTPAP are positively correlated with patient survival, with (Fig. 2A, ii) showing COX15 as an example. Supplementary Fig. S1A and S1B (i) shows similar plots with TFAM and MTPAP mRNA levels. The MAOB:mitochondria (COX15) ratio represents a risk factor for MP-Pt(IV) toxicity and patients with high MAOB:COX15 ratios have very poor survival (Fig. 2A, iii). High MAOB:mitochondria ratios, estimated using proxies TFAM and MTPAP, replicate this result (Supplementary Fig. S1A and S1B, ii).

Like MAOB, SLC2A10 levels are also highly correlated to poor patient outcomes (Fig. 2B, i) and high SLC2A10/COX15 ratios correlate with poor outcome (Fig. 2B, ii).

LDHA expression levels are known to be correlated with the Warburg shift in cancers, including GBM (42), and we observed that higher the Warburg shift, the poorer the GBM patient outcome (Supplementary Fig. S1C, i). A better measure of the Warburg shift is the LDHA:COX15 ratio, which should reflect the relative fluxes of anaerobic to aerobic respiration (Supplementary Fig. S1C, ii). As expected, the greater the Warburg shift, the poorer the patient outcome.

The aggressive GBM phenotype (high MAOB, high SLC2A10, and low mitochondria) is likely to be sensitive to MP-Pt(IV). In contrast, none of the other tissues in the body have this phenotype. The levels of MAOB and mitochondrial proxies in a subset of normal human tissues are presented in Supplementary Fig. S2. Data from the GTEx database (https://gtexportal.org/home/) show that MAOB levels tightly track mitochondrial proxies in normal tissues. As the MAOB:mitochondria ratio is >15 in GBM compared with NHA (17), neither liver nor brain should generate and accumulate P+-Pt(IV) better than GBM.

Oxidation of MP-Pt(IV) by rhMAOA and rhMAOB

Enzymatic studies of MP-Pt(IV) with both rhMAOA and rhMAOB were examined using two methods, the Amplex Red peroxide assay and UV spectroscopy. MAOB kinetics as a function of [MP-Pt(IV)] are shown in Fig. 3A. The fitted Michaelis–Menten plot indicates that the Km of MAOB for MP-Pt(IV) is 210 μmol/L and the Vmax was 13% of that observed using saturating benzylamine.

Figure 3.

A, Michaelis–Menten kinetic plot of rhMAOB generation of hydrogen peroxide, measured by Amplex red/HRP. Each point represents the mean of n = 3 different individual experiments. MAOA gave no activity. Kinetic modeling shows the Km is approximately 210 μmol/L. B, Kinetics of UV spectral changes of the oxidation of MP-Pt(IV) by MAOB and MAOA. Scans were taken at 5, 15, 30, 60, 90, 120, and 150 minutes and the difference spectra shows the loss of the tetrahydropyridine ring (trough at 292 nm), and the appearance of the dihydropyridinium ring (peak 315 nm), followed by the formation of the P+-Pt(IV) mitochondrial-targeting final product (peak 250 nm). No such optical changes were observed using rhMAOA.

Figure 3.

A, Michaelis–Menten kinetic plot of rhMAOB generation of hydrogen peroxide, measured by Amplex red/HRP. Each point represents the mean of n = 3 different individual experiments. MAOA gave no activity. Kinetic modeling shows the Km is approximately 210 μmol/L. B, Kinetics of UV spectral changes of the oxidation of MP-Pt(IV) by MAOB and MAOA. Scans were taken at 5, 15, 30, 60, 90, 120, and 150 minutes and the difference spectra shows the loss of the tetrahydropyridine ring (trough at 292 nm), and the appearance of the dihydropyridinium ring (peak 315 nm), followed by the formation of the P+-Pt(IV) mitochondrial-targeting final product (peak 250 nm). No such optical changes were observed using rhMAOA.

Close modal

The enzyme catalyzed consumption of the MP-species and appearance of the corresponding dihydropyridinium and pyridinium species were studied using UV spectroscopy (21) to demonstrate the oxidation of MP-Pt(IV) to P+-Pt(IV) via the DP-Pt(IV) (Fig. 3B; ref. 43). We observed no activity of MAOA with MP-Pt(IV) in either assay system. To ensure that the rhMAOA we used was catalytically active, we successfully performed validation control studies using the oxidation of tyramine and benzylamine (21) as substrates. These studies prove that MP-Pt(IV) is selectively oxidized by MAOB. Additional control was performed using the MAOB-specific inhibitor, selegiline, which abolished MAOB catalyzed MP-Pt(IV) oxidation.

Reduction of Pt(IV) by ascorbate

Experiments conducted to verify the reduction of MP-Pt(IV) by ascorbate into cisplatin are detailed in Supplementary Fig. S3. The products of the reduction of MP-Pt(IV) by ascorbate were examined using mass spectroscopy (Supplementary Fig. S3A). MP-Pt(IV) reduction occurs by a pair of one-electron reduction steps, the first step displacing the axial chloride and forming the Pt(III) species, and the second step releasing the axial MP-ethylene glycol and cisplatin (Supplementary Fig. S3B). Optical studies using 5 mmol/L ascorbate with 100 μmol/L MP-Pt(IV) indicate the reduction of MP-Pt(IV) was pseudo first-order (Supplementary Fig. S3C,i and ii).

In vitro toxicity in primary GBM cultures

The cytotoxic effect of MP-Pt(IV) is affected by the generation of cisplatin within the mitochondrial matrix, and this conversion should be accelerated by increasing the concentration of mitochondrial ascorbate. The rates at which different Pt(IV) compounds are reduced into their Pt(II) daughter drugs correlate with their cytotoxic effect against cancer cells (44), with ascorbate as the main biological reductant (45).

Figure 4 shows that MP-Pt(IV) is toxic toward both GBM115 and GBM157 cells with IC50s of approximately 6 μmol/L and approximately 3 μmol/L, respectively. Dehydroascorbate sensitized cells toward MP-Pt(IV) toxicity, whereas ascorbate provided some protection toward MP-Pt(IV), with significance being found at and beyond 8 and 4 μmol/L, respectively (P < 0.05), compared with cells preincubated with unsupplemented media. Dehydroascorbate is imported into GBM cells via hexose transporters and elevates ascorbate. GBM cells do not typically import ascorbate, as they express low/no levels of the Na+/ascorbate cotransporters, SVCT1&2. Ascorbate added to the media of cultured cells typically acts as a prooxidant due to the generation of hydrogen peroxide resulting from ascorbate auto-oxidation (46), and so paradoxically externally added ascorbate can depress intracellular ascorbate levels.

Figure 4.

Toxicity of MP-Pt(IV) in two different GBM primary cultures preincubated overnight with dehydroascorbate (DHA, 5 mmol/L) or ascorbate (Asc, 5 mmol/L) or media alone. After media replacement, the cells were incubated with MP-Pt(IV) for 24 hours, then incubated with Hoechst 33342 nucleic acid stain, fixed with paraformaldehyde, and fluorescence images were taken so that living cells could be counted. In both cases, preincubation of GBM cells with dehydroascorbate sensitized them to MP-Pt(IV), whereas ascorbate desensitized them. Mean ± SD, n = 4. t Test of cumulative means shows when cell numbers diverge from media control cells at P < 0.05 level.

Figure 4.

Toxicity of MP-Pt(IV) in two different GBM primary cultures preincubated overnight with dehydroascorbate (DHA, 5 mmol/L) or ascorbate (Asc, 5 mmol/L) or media alone. After media replacement, the cells were incubated with MP-Pt(IV) for 24 hours, then incubated with Hoechst 33342 nucleic acid stain, fixed with paraformaldehyde, and fluorescence images were taken so that living cells could be counted. In both cases, preincubation of GBM cells with dehydroascorbate sensitized them to MP-Pt(IV), whereas ascorbate desensitized them. Mean ± SD, n = 4. t Test of cumulative means shows when cell numbers diverge from media control cells at P < 0.05 level.

Close modal

Additional control experiments established that blocking MAOB activity with 10 μmol/L selegiline preincubation completely protected them from incubation with 14 μmol/L MP-Pt(IV). Growth experiments conducted with both GBM cultures incubated with the precursor Pt(IV)-ethylene glycol demonstrated no change in cell numbers.

To investigate the mechanism of MP-Pt(IV) toxicity, we incubated GBM157 cells with or without 4 μmol/L MP-Pt(IV) for 24 hours and examined markers of ROS and mitochondria function. The ROS probes, H2-DCF-AM and MitoSox, indicate that MP-Pt(IV) causes an elevation in the steady-state levels of cellular peroxide and mitochondrial matrix superoxide, respectively (Fig. 5A and B).

Figure 5.

A, GBM157 cells surviving 24 hours of treatment with 4 μmol/L MP-Pt(IV) have slightly elevated cellular peroxide levels, as measured by DCF formed. Statistically significant increases were observed in mitochondrial superoxide (B) and caspase-3 (C), both markers of mitochondrial stress. In both cases, the elevation was greatest in cells preincubated with dehydroascorbate (DHA). Preincubation with both ascorbate (Asc) and dehydroascorbate also elevated caspase-3 activity in cells untreated with MP-Pt(IV). MP-Pt(IV) also causes upregulation of mitochondria in surviving GBM157 cells, with cells surviving 24 hours of treatment with 4 μmol/L MP-Pt(IV) increasing both mitochondrial numbers (measured by MitoTracker levels per cell) and MAOB (D and E). Experiments were performed at n = 8 (A–C) and n = 4 (allowing n = 4 unlabeled background controls; D and E), statistical analysis using t test.

Figure 5.

A, GBM157 cells surviving 24 hours of treatment with 4 μmol/L MP-Pt(IV) have slightly elevated cellular peroxide levels, as measured by DCF formed. Statistically significant increases were observed in mitochondrial superoxide (B) and caspase-3 (C), both markers of mitochondrial stress. In both cases, the elevation was greatest in cells preincubated with dehydroascorbate (DHA). Preincubation with both ascorbate (Asc) and dehydroascorbate also elevated caspase-3 activity in cells untreated with MP-Pt(IV). MP-Pt(IV) also causes upregulation of mitochondria in surviving GBM157 cells, with cells surviving 24 hours of treatment with 4 μmol/L MP-Pt(IV) increasing both mitochondrial numbers (measured by MitoTracker levels per cell) and MAOB (D and E). Experiments were performed at n = 8 (A–C) and n = 4 (allowing n = 4 unlabeled background controls; D and E), statistical analysis using t test.

Close modal

Treatment with MP-Pt(IV) caused a modest increase in cellular peroxide levels, which was not statistically significant in any of the individual groups, but was statistically significant when the data from the three groups were pooled (P < 0.01). MP-Pt(IV) caused a 2-fold elevation of superoxide levels and such elevation typically lowers mitochondrial ΔΨ by inhibiting aconitase (47, 48) and also by activating phospholipase A2 (providing free fatty acids for uncoupling protein; ref. 49). In conditions where ΔΨ is low and ROS is high, mitochondria can undergo the permeability transition, releasing cytochrome c and Smac/DIABLO into the cytosol, thereby initiating the caspase-3 apoptotic cascade. Using a caspase-3–specific substrate, we found that MP-Pt(IV) causes the activation of this apoptotic cascade, and cells preincubated with dehydroascorbate had a much greater increase in caspase-3 activation (Fig. 5C).

In GBM157 cells that survived 4 μmol/L MP-Pt(IV), we found upregulation in mitochondrial numbers per surviving cell using the probe, MitoTracker (Fig. 5D). The degree of upregulation was affected by preincubation, dehydroascorbate > control > ascorbate. MAOB levels were also upregulated, again with dehydroascorbate > control > ascorbate (Fig. 5E). In Supplementary Fig. S4, we show representative images of GBM115 and GBM157 cells treated with increasing concentrations of MP-Pt(IV) labeled with MitoTracker and for MAOB, with nuclei labeled with DAPI. MAOB levels increased following incubation with MP-Pt(IV) and mitochondrial levels responded to this prodrug. Of note was the change in cellular morphology that resulted from treatment and the swollen, distended nuclei. This upregulation of mitochondria and MAOB, in response to MP-Pt(IV) treatment, was also previously seen with MP-MUS (21, 24).

Intracranial model of primary GBM

We wanted to determine whether the addition of MP-Pt(IV) improved the efficacy of the Stupp protocol. Figure 6 shows the survival of mice with intracranial primary GBM xenografts treated with MP-Pt(IV) and/or conventional chemoradiotherapy. Forty-eight mice were given xenografts and, on day 8, the 42 heaviest mice were randomized into seven groups of 6 animals each.

Figure 6.

The survival of seven groups of mice, n = 6, treated with combinations/permutations of temozolomide (TMZ) and/or MP-Pt(IV) chemoradiation. A, Animal survival using the traditional Kaplan–Meier survival curve format, without radiation (IR) (top) and with radiation (bottom). B, The average mouse survival days from day 1 after xenografting versus study day 1. The curves are color coded, and the treatment given is numbered #1 to #7 (key in the right). Treatments were given on days 11, 13, 15, 18, 20, and 22 after xenografting, indicated by red arrows. The best outcome was observed in mice treated with temozolomide and MP-Pt(IV) without radiation (#4), where all mice survived till the end of the study (day 300), but there is no statistical difference between temozolomide and MP-Pt(IV) ± radiation. Statistical significance of the four MP-Pt(IV) treatments, with respect to control, are all P < 0.0001.

Figure 6.

The survival of seven groups of mice, n = 6, treated with combinations/permutations of temozolomide (TMZ) and/or MP-Pt(IV) chemoradiation. A, Animal survival using the traditional Kaplan–Meier survival curve format, without radiation (IR) (top) and with radiation (bottom). B, The average mouse survival days from day 1 after xenografting versus study day 1. The curves are color coded, and the treatment given is numbered #1 to #7 (key in the right). Treatments were given on days 11, 13, 15, 18, 20, and 22 after xenografting, indicated by red arrows. The best outcome was observed in mice treated with temozolomide and MP-Pt(IV) without radiation (#4), where all mice survived till the end of the study (day 300), but there is no statistical difference between temozolomide and MP-Pt(IV) ± radiation. Statistical significance of the four MP-Pt(IV) treatments, with respect to control, are all P < 0.0001.

Close modal

Treatments began on day 11, and each animal was given six treatments in all, on days 11, 13, 15, 18, 20, and 22. The groups underwent combination/permutations of three treatments (Monday, Wednesday, and Friday):

  • Tail vein injection of 15 mg/kg MP-Pt(IV) (≡8.2 mg/kg cisplatin) in saline or saline.

  • Temozolomide 5 mg/kg, given by gavage or oral gavage solution.

  • Whole-head radiation (2 Gy from cesium source) or sham radiation.

For comparison, the clinical doses for temozolomide, during chemoradiation, were 150 mg/m2 (∼33.8 mg/kg dose in mice) and for cisplatin, 75 mg/m2 (∼16.9 mg/kg dose in mice).

The animals were monitored daily for the length of the study and were sacrificed when they reached an ethical endpoint. The study was terminated on day 300, as beyond this point the nude mice began to suffer age-dependent maladies.

The animal weights (Supplementary Fig. S5) indicate that MP-Pt(IV) was well-tolerated and that animals gained weight after treatment.

Figure 6A shows animal survival using the traditional Kaplan–Meier survival curve format, with treatment groups that received sham radiation in the top plot and those that received six cycles of 2 Gy whole-head radiation shown in the bottom plot. Figure 6B shows a different presentation of the survival data, where we plotted the average mouse survival days post-xenograft versus study day. The curves are color coded and are shown in key form in the right panel.

In mice that were not irradiated (Fig. 6A, top), it was clear that temozolomide and MP-Pt(IV) potentiated each other's anticancer effect, but did not cause any drop in animal's weight (a metric of health). Combining temozolomide and MP-Pt(IV) provided immortalization for 10 months and GBM tumors could not be identified in brain tissue at the studies end. When given as a monotherapy, MP-Pt(IV) proved highly effective, with 50% of animals surviving 150 days postimplantation. The combined therapy of MP-Pt(IV) with Stupp was far more effective than Stupp alone (P < 0.0001), with only 1 animal reaching a tumor-related ethical endpoint and another suffering from an ulcer at an injection point. Detailed statistical analysis of the different treatment arms compared with control arm and cumulative mouse survival ratios of different treatment and control arms are shown in Supplementary Table S1.

Lipophilic cations such as triphenylphosphonium, which rapidly accumulate in the mitochondrial matrix, have been explored for mitochondrial-targeting of anticancer drug conjugates (23). Our approach exploits the overexpressed enzyme, MAOB, in the GBM mitochondria for an in situ generation of a lipophilic cation. Both MAOA and MAOB are recognized to mediate and regulate intracellular signaling pathways involved in neuronal growth (50). MAOA has been linked to developing neuronal architecture and synaptic activity, and with maladies like the onset of psychiatric disorders, depression, and aggressive impulsive behavior. In contrast, MAOB has been associated with diseases involving neuronal loss such as Alzheimer and Parkinson diseases. Our drug design strategy exploits the biochemical function of MAOB to enable mitochondrial targeting of a prodrug in GBM.

An ideal anticancer drug is highly toxic toward cancer cells, but is nontoxic to normal cells. In practice, however, most chemotherapeutics only show differential toxicity: highly toxic toward cancer cells, but only slightly less toxic toward the next most sensitive tissue(s). We examined the cancer cell type, especially the most aggressive phenotype, to identify enzyme system(s) that are highly upregulated, compared with the other normal cells, which could then be used to catalyze the maturation of a nontoxic prodrug into a toxic drug. GBMs, like many cancers, have low levels of mitochondria and yet simultaneously show high levels of the mitochondrial enzyme, MAOB, and the mitochondrial dehydroascorbate transporter, Glut10. The prodrug, MP-Pt(IV), was thus designed to exploit high mitochondrial MAOB and Glut10 to generate cisplatin within the mitochondria of GBM.

Within the mitochondria, the Pt(IV) group is activated by ascorbate/GSH reduction to give rise to cisplatin, within the mitochondrial matrix. Compared with our first-generation prodrug, MP-MUS (24), it is 10-fold more efficacious toward GBM in vitro and in vivo and can be used at much higher concentrations (15 compared with 8 mg/kg for MP-MUS) because of higher aqueous solubility (≈8 mg/mL in water compared with 1 mg/mL in 40% Kolliphor in saline for MP-MUS) and stability.

Data mining demonstrated that the most aggressive GBM shows a pronounced Warburg shift, which is linked to elevated MAOB and Glut10 levels, and low levels of mitochondria within these cancer cells. The mitochondrial damage inflicted by MP-Pt(IV) is a function of MAOB and Glut10 levels, both of which aid the generation of cisplatin from P+-Pt(IV) within the mitochondrial matrix.

GBM cells are acutely sensitive to MP-Pt(IV), with MP-Pt(IV)–treated cultured cells displaying elevated mitochondrial superoxide/peroxide production and the release cytochrome c, activating caspase-3, and priming the affected cells for apoptosis. GBM cells that are exposed to nonlethal MP-Pt(IV) treatment respond by upregulating mitochondrial levels, which also lead to an increase in the levels of MAOB, and thereby increasing their sensitivity to subsequent treatments with MP-Pt(IV).

MP-Pt(IV) monotherapy is highly effective in intracranial models of GBM, but when used in conjunction with temozolomide, it is curative. Animals treated with MP-Pt(IV) were healthy and gained weight during their treatment course. This potentiation of temozolomide therapy means that MP-Pt(IV) has a high potential for integration with the Stupp protocol. These studies demonstrate that adding MP-Pt(IV) to a chemoradiotherapy protocol could address a critical need in GBM treatment and makes MP-Pt(IV) an excellent candidate for preclinical development.

D.S. Baskin reports a patent submitted for this work (pending). No commercial activities have occurred or are anticipated any time soon. This work needs further development. No potential conflicts of interest were disclosed by the other authors.

S. Raghavan: Conceptualization, data curation, investigation, methodology, writing-original draft, writing-review and editing. D.S. Baskin: Conceptualization, resources, supervision, funding acquisition, methodology, project administration, writing-review and editing. M.A. Sharpe: Conceptualization, data curation, software, formal analysis, supervision, validation, investigation, visualization, methodology, writing-original draft, writing-review and editing.

This work was supported by Donna and Kenneth Peak, The Kenneth R. Peak Foundation, The John S. Dunn Foundation, The Taub Foundation, The Blanche Green Fund of the Pauline Sterne Wolff Memorial Foundation, The Kelly Kicking Cancer Foundation, The Gary and Marlee Schwarz Foundation, and The Methodist Hospital Foundation & The Veralan Foundation. The John S. Dunn Foundation also supports M.A. Sharpe's distinguished professorship. We are grateful to the many patients and their families who have participated in our studies, and who are dedicated to joining us in our fight against brain cancer. We thank Tanvi Kumar and Sophie Lopez for their technical support. The results shown here are in whole or partly based upon data generated by TCGA Research Network (https://www.cancer.gov/tcga. CCGA), and Gravendeel databases were accessed via GlioVis (http://gliovis.bioinfo.cnio.es/).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Chinot
OL
,
Wick
W
,
Mason
W
,
Henriksson
R
,
Saran
F
,
Nishikawa
R
, et al
Bevacizumab plus radiotherapy-temozolomide for newly diagnosed glioblastoma
.
New Engl J Med
2014
;
370
:
709
22
.
2.
Stupp
R
,
Mason
WP
,
van den Bent
MJ
,
Weller
M
,
Fisher
B
,
Taphoorn
MJB
, et al
Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma
.
New Engl J Med
2005
;
352
:
987
96
.
3.
Herrlinger
U
,
Tzaridis
T
,
Mack
F
,
Steinbach
JP
,
Schlegel
U
,
Sabel
M
, et al
Lomustine-temozolomide combination therapy versus standard temozolomide therapy in patients with newly diagnosed glioblastoma with methylated MGMT promoter (CeTeG/NOA-09): a randomised, open-label, phase 3 trial
.
Lancet
2019
;
393
:
678
88
.
4.
Ballo
MT
,
Urman
N
,
Lavy-Shahaf
G
,
Grewal
J
,
Bomzon
Z
,
Toms
S
. 
Correlation of tumor treating fields dosimetry to survival outcomes in newly diagnosed glioblastoma: a large-scale numerical simulation-based analysis of data from the phase 3 EF-14 randomized trial
.
Int J Radiat Oncol Biol Phys
2019
;
104
:
1106
13
.
5.
Fecci
PE
,
Sampson
JH
. 
The current state of immunotherapy for gliomas: an eye toward the future
.
J Neurosurg
2019
;
131
:
657
.
6.
Yang
L
,
Lin
C
,
Wang
L
,
Guo
H
,
Wang
X
. 
Hypoxia and hypoxia-inducible factors in glioblastoma multiforme progression and therapeutic implications
.
Exp Cell Res
2012
;
318
:
2417
26
.
7.
Sharpe
MA
,
Ismail
N
,
Baskin
DS
. 
Metabolic sculpting of the mitochondria, cell signaling and the cancer phenotype
.
Transl Cancer Res
2017:
6.
8.
Kucharzewska
P
,
Christianson
HC
,
Belting
M
. 
Global profiling of metabolic adaptation to hypoxic stress in human glioblastoma cells
.
PLoS One
2015
;
10
:
e0116740
.
9.
Chinopoulos
C
,
Seyfried
TN
. 
Mitochondrial substrate-level phosphorylation as energy source for glioblastoma: review and hypothesis
.
ASN Neuro
2018
;
10
:
1759091418818261
.
10.
Cantoni
O
,
Guidarelli
A
,
Fiorani
M
. 
Mitochondrial uptake and accumulation of vitamin C: what can we learn from cell culture studies?
Antioxid Redox Signal
2017
;
29
:
1502
15
.
11.
Lee
YC
,
Huang
HY
,
Chang
CJ
,
Cheng
CH
,
Chen
YT
. 
Mitochondrial GLUT10 facilitates dehydroascorbic acid import and protects cells against oxidative stress: mechanistic insight into arterial tortuosity syndrome
.
Hum Mol Genet
2010
;
19
:
3721
33
.
12.
Le Calvé
B
,
Rynkowski
M
,
Le Mercier
M
,
Bruyère
C
,
Lonez
C
,
Gras
T
, et al
Long-term in vitro treatment of human glioblastoma cells with temozolomide increases resistance in vivo through up-regulation of GLUT transporter and aldo-keto reductase enzyme AKR1C expression
.
Neoplasia
2010
;
12
:
727
39
.
13.
Sharpe
MA
,
Raghavan
S
,
Baskin
DS
. 
PAM-OBG: a monoamine oxidase B specific prodrug that inhibits MGMT and generates DNA interstrand crosslinks, potentiating temozolomide and chemoradiation therapy in intracranial glioblastoma
.
Oncotarget
2018
;
9
:
23923
43
.
14.
Kalyanaraman
B
,
Cheng
G
,
Hardy
M
,
Ouari
O
,
Lopez
M
,
Joseph
J
, et al
A review of the basics of mitochondrial bioenergetics, metabolism, and related signaling pathways in cancer cells: therapeutic targeting of tumor mitochondria with lipophilic cationic compounds
.
Redox Biol
2018
;
14
:
316
27
.
15.
Gabilondo
AM
,
Hostalot
C
,
Garibi
JM
,
Meana
JJ
,
Callado
LF
. 
Monoamine oxidase B activity is increased in human gliomas
.
Neurochem Int
2008
;
52
:
230
4
.
16.
Gerber
NK
,
Goenka
A
,
Turcan
S
,
Reyngold
M
,
Makarov
V
,
Kannan
K
, et al
Transcriptional diversity of long-term glioblastoma survivors
.
Neuro Oncol
2014
;
16
:
1186
95
.
17.
Sharpe
MA
,
Baskin
DS
. 
Monoamine oxidase B levels are highly expressed in human gliomas and are correlated with the expression of HiF-1α and with transcription factors Sp1 and Sp3
.
Oncotarget
2016
;
7
:
3379
93
.
18.
Su
T
,
Liu
P
,
Ti
X
,
Wu
S
,
Xue
X
,
Wang
Z
, et al
HIF1α, EGR1 and SP1 co-regulate the erythropoietin receptor expression under hypoxia: an essential role in the growth of non-small cell lung cancer cells
.
Cell Commun Signal
2019
;
17
:
152
.
19.
Sapetschnig
A
,
Koch
F
,
Rischitor
G
,
Mennenga
T
,
Suske
G
. 
Complexity of translationally controlled transcription factor Sp3 isoform expression
.
J Biol Chem
2004
;
279
:
42095
105
.
20.
Discher
DJ
,
Bishopric
NH
,
Wu
X
,
Peterson
CA
,
Webster
KA
. 
Hypoxia regulates beta-enolase and pyruvate kinase-M promoters by modulating Sp1/Sp3 binding to a conserved GC element
.
J Biol Chem
1998
;
273
:
26087
93
.
21.
Sharpe
MA
,
Han
J
,
Baskin
AM
,
Baskin
DS
. 
Design and synthesis of a MAO-B-selectively activated prodrug based on MPTP: a mitochondria-targeting chemotherapeutic agent for treatment of human malignant gliomas
.
ChemMedChem
2015
;
10
:
621
8
.
22.
Ross
MF
,
Kelso
GF
,
Blaikie
FH
,
James
AM
,
Cocheme
HM
,
Filipovska
A
, et al
Lipophilic triphenylphosphonium cations as tools in mitochondrial bioenergetics and free radical biology
.
Biochemistry (Mosc)
2005
;
70
:
222
30
.
23.
Murphy
MP
. 
Targeting lipophilic cations to mitochondria
.
Biochim Biophys Acta
2008
;
1777
:
1028
31
.
24.
Sharpe
MA
,
Livingston
AD
,
Gist
TL
,
Ghosh
P
,
Han
J
,
Baskin
DS
. 
Successful treatment of intracranial glioblastoma xenografts with a monoamine oxidase B-activated pro-drug
.
EBioMedicine
2015
;
2
:
1122
32
.
25.
Ravera
M
,
Gabano
E
,
Pelosi
G
,
Fregonese
F
,
Tinello
S
,
Osella
D
. 
A new entry to asymmetric platinum(IV) complexes via oxidative chlorination
.
Inorg Chem
2014
;
53
:
9326
35
.
26.
Jovanovic
S
,
Petrovic
B
,
Bugarcic
ZD
,
van Eldik
R
. 
Reduction of some Pt(IV) complexes with biologically important sulfur-donor ligands
.
Dalton Trans
2013
;
42
:
8890
6
.
27.
Lemma
K
,
Berglund
J
,
Farrell
N
,
Elding
LI
. 
Kinetics and mechanism for reduction of anticancer-active tetrachloroam(m)ine platinum(IV) compounds by glutathione
.
J Biol Inorg Chem
2000
;
5
:
300
6
.
28.
Wang
Z
,
Deng
Z
,
Zhu
G
. 
Emerging platinum(iv) prodrugs to combat cisplatin resistance: from isolated cancer cells to tumor microenvironment
.
Dalton Trans
2019
;
48
:
2536
44
.
29.
Marí
M
,
Morales
A
,
Colell
A
,
García-Ruiz
C
,
Fernández-Checa
JC
. 
Mitochondrial glutathione, a key survival antioxidant
.
Antioxid Redox Signal
2009
;
11
:
2685
700
.
30.
Bansal
A
,
Simon
MC
. 
Glutathione metabolism in cancer progression and treatment resistance
.
J Cell Biol
2018
;
217
:
2291
8
.
31.
Saura
J
,
Nadal
E
,
van den Berg
B
,
Vila
M
,
Bombi
JA
,
Mahy
N
. 
Localization of monoamine oxidases in human peripheral tissues
.
Life Sci
1996
;
59
:
1341
9
.
32.
Chen
K
. 
Organization of MAO A and MAO B promoters and regulation of gene expression
.
Neurotoxicology
2004
;
25
:
31
6
.
33.
Segade
F
,
Allred
DC
,
Bowden
DW
. 
Functional characterization of the promoter of the human glucose transporter 10 gene
.
Biochim Biophys Acta Gene Struct Expression
2005
;
1730
:
147
58
.
34.
Petruzzella
V
,
Tiranti
V
,
Fernandez
P
,
Ianna
P
,
Carrozzo
R
,
Zeviani
M
. 
Identification and characterization of human cDNAs specific to BCS1, PET112, SCO1, COX15, and COX11, five genes involved in the formation and function of the mitochondrial respiratory chain
.
Genomics
1998
;
54
:
494
504
.
35.
Oquendo
CE
,
Antonicka
H
,
Shoubridge
EA
,
Reardon
W
,
Brown
GK
. 
Functional and genetic studies demonstrate that mutation in the COX15 gene can cause Leigh syndrome
.
J Med Genet
2004
;
41
:
540
.
36.
Araujo
LF
,
Siena
ADD
,
Plaça
JR
,
Brotto
DB
,
Barros
II
,
Muys
BR
, et al
Mitochondrial transcription factor A (TFAM) shapes metabolic and invasion gene signatures in melanoma
.
Sci Rep
2018
;
8
:
14190
.
37.
Slomovic
S
,
Schuster
G
. 
Stable PNPase RNAi silencing: its effect on the processing and adenylation of human mitochondrial RNA
.
RNA
2008
;
14
:
310
23
.
38.
Wilson
WC
,
Hornig-Do
HT
,
Bruni
F
,
Chang
JH
,
Jourdain
AA
,
Martinou
JC
, et al
A human mitochondrial poly(A) polymerase mutation reveals the complexities of post-transcriptional mitochondrial gene expression
.
Hum Mol Genet
2014
;
23
:
6345
55
.
39.
Reznik
E
,
Miller
ML
,
Şenbabaoğlu
Y
,
Riaz
N
,
Sarungbam
J
,
Tickoo
SK
, et al
Mitochondrial DNA copy number variation across human cancers
.
Elife
2016
;
5
:
e10769
.
40.
Zhao
B
,
Ranguelova
K
,
Jiang
J
,
Mason
RP
. 
Studies on the photosensitized reduction of resorufin and implications for the detection of oxidative stress with Amplex Red
.
Free Radic Biol Med
2011
;
51
:
153
9
.
41.
Iwami
K
,
Momota
H
,
Natsume
A
,
Kinjo
S
,
Nagatani
T
,
Wakabayashi
T
. 
A novel method of intracranial injection via the postglenoid foramen for brain tumor mouse models
.
J Neurosurg
2012
;
116
:
630
5
.
42.
Di
H
,
Zhang
X
,
Guo
Y
,
Shi
Y
,
Fang
C
,
Yuan
Y
, et al
Silencing LDHA inhibits proliferation, induces apoptosis and increases chemosensitivity to temozolomide in glioma cells
.
Oncol Lett
2018
;
15
:
5131
6
.
43.
Fritz
RR
,
Abell
CW
,
Patel
NT
,
Gessner
W
,
Brossi
A
. 
Metabolism of the neurotoxin in MPTP by human liver monoamine oxidase B
.
FEBS Lett
1985
;
186
:
224
8
.
44.
Göschl
S
,
Schreiber-Brynzak
E
,
Pichler
V
,
Cseh
K
,
Heffeter
P
,
Jungwirth
U
, et al
Comparative studies of oxaliplatin-based platinum(iv) complexes in different in vitro and in vivo tumor models
.
Metallomics
2017
;
9
:
309
22
.
45.
Sinisi
M
,
Intini
FP
,
Natile
G
. 
Dependence of the reduction products of platinum(IV) prodrugs upon the configuration of the substrate, bulk of the carrier ligands, and nature of the reducing agent
.
Inorg Chem
2012
;
51
:
9694
704
.
46.
Chen
Q
,
Espey
MG
,
Sun
AY
,
Lee
JH
,
Krishna
MC
,
Shacter
E
, et al
Ascorbate in pharmacologic concentrations selectively generates ascorbate radical and hydrogen peroxide in extracellular fluid in vivo
.
Proc Natl Acad Sci U S A
2007
;
104
:
8749
54
.
47.
Lushchak
OV
,
Piroddi
M
,
Galli
F
,
Lushchak
VI
. 
Aconitase post-translational modification as a key in linkage between Krebs cycle, iron homeostasis, redox signaling, and metabolism of reactive oxygen species
.
Redox Rep
2014
;
19
:
8
15
.
48.
Armstrong
JS
,
Whiteman
M
,
Yang
H
,
Jones
DP
. 
The redox regulation of intermediary metabolism by a superoxide-aconitase rheostat
.
Bioessays
2004
;
26
:
894
900
.
49.
Jaburek
M
,
Jezek
J
,
Zelenka
J
,
Jezek
P
. 
Antioxidant activity by a synergy of redox-sensitive mitochondrial phospholipase A2 and uncoupling protein-2 in lung and spleen
.
Int J Biochem Cell Biol
2013
;
45
:
816
25
.
50.
Shih
JC
,
Chen
K
,
Ridd
MJ
. 
Monoamine oxidase: from genes to behavior
.
Annu Rev Neurosci
1999
;
22
:
197
217
.