Redox-Active Mn Porphyrin-based Potent SOD Mimic, MnTnBuOE-2-PyP(5+), Enhances Carbenoxolone-Mediated TRAIL-Induced Apoptosis in Glioblastoma Multiforme

Abstract

Glioblastoma multiforme is the most malignant tumor of the brain and is challenging to treat due to its highly invasive nature and heterogeneity. Malignant brain tumor displays high metabolic activity which perturbs its redox environment and in turn translates to high oxidative stress. Thus, pushing the oxidative stress level to achieve the maximum tolerable threshold that induces cell death is a potential strategy for cancer therapy. Previously, we have shown that gap junction inhibitor, carbenoxolone (CBX), is capable of enhancing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) -induced apoptosis in glioma cells. Since CBX is known to induce oxidative stress, we hypothesized that the addition of another potent mediator of oxidative stress, powerful SOD mimic MnTnBuOE-2-PyP(5+) (MnBuOE), could further enhance TRAIL-driven therapeutic efficacy in glioma cells. Our results showed that combining TRAIL + CBX with MnBuOE significantly enhances cell death of glioma cell lines and this enhancement could be further potentiated by CBX pretreatment. MnBuOE-driven cytotoxicity is due to its ability to take advantage of oxidative stress imposed by CBX + TRAIL system, and enhance it in the presence of endogenous reductants, ascorbate and thiol, thereby producing cytotoxic H2O2, and in turn inducing death of glioma cells but not normal astrocytes. Most importantly, combination treatment significantly reduces viability of TRAIL-resistant Asian patient-derived glioma cells, thus demonstrating the potential clinical use of our therapeutic system. It was reported that H2O2 is involved in membrane depolarization-based sensitization of cancer cells toward TRAIL. MnBuOE is entering Clinical Trials as a normal brain radioprotector in glioma patients at Duke University increasing Clinical relevance of our studies.

Keywords: Ascorbate; Carbenoxolone; Glioma; Manganese porphyrin; NAC; SOD mimic; TRAIL-modified human mesenchymal stem cells.

Fig. 1

MnBuOE is non-cytotoxic and enhanced CBX-mediated TRAIL-induced cell death in human glioma. a The effect of various concentrations of MnBuOE on ΔGli36 human glioma cells and immortalized normal human astrocytes (iNHA) was examined at 48 h. At the desired time point, CCK-8 assay was performed to determine the cell viability. b ΔGli36 glioma cells were treated with different dose of MnBuOE in presence of TRAIL and 100 μM of CBX. CCK-8 assay was then performed at 48 h to determine the effect of treatment on the cells’ viability. **, p < 0.01; ***, p < 0.001

Fig. 2

Triple combination of TRAIL, CBX and MnBuOE conferred better efficacy in human gliomas than single or double treatment. a ΔGli36 glioma cells and b Immortalized normal human astrocytes, iNHA cells were subjected to TRAIL, (i) 100 μM CBX or (ii) GZA, as well as 50 μM MnBuOE for 48 h and 72 h. At the desired time points, cell viability was determined with CCK-8 assay. n.s, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001

Fig. 3

Pre-incubation with CBX enhanced the efficacy of the triple combination in human glioma. ΔGli36 human glioma cells were subjected to either a 24 h pre-incubation or b no pre-incubation with 100 μM CBX or GZA prior to be treated in combination with TRAIL and 25 μM of MnBuOE. Following 48 h of combination treatment, viability of the cells was determined with CCK-8 assay. n.s, p > 0.05; **, p < 0.01; ***, p < 0.001

Fig. 4

Enhanced efficacy of triple combination of TRAIL, CBX and MnBuOE was also seen in U87MG human glioma cells but not iNHA. a U87MG glioma cells and b iNHA cells were pre-incubated with 100 μM of CBX or GZA for 24 h. Cells were then subjected to combined treatment of TRAIL, CBX and MnBuOE for 48 h, followed by CCK-8 cell viability assay. n.s, p > 0.05; **, p < 0.01; ***, p < 0.001

Fig. 5

Triple combination could enhance TRAIL-induced apoptosis in resistant Asian primary GBM. a Short-term culture of Asian primary glioma cells was exposed to TRAIL for 72 h. Cell death was determined with trypan blue dye exclusion assay. Asian primary glioma cells, b GBM8401, c GBM8901, d G5T/VGH, and e NNI23 were pre-incubated with 100 μM of CBX or GZA for 24 h. Cells were then treated with combination treatment of TRAIL, CBX and MnBuOE for 48 h, followed by CCK-8 cell viability assay. n.s, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001

Fig. 6

Enhancement of TRAIL-induced apoptosis by triple combination treatment in presence of cellular reductants. ΔGli36 human glioma cells were pre-incubated with 100 μM of CBX for 24 h. Cells were then incubated with a 10 mM NAC for 1 h followed by combination treatment of TRAIL, 100 μM of CBX and 25 μM of MnBuOE or b combination treatment in presence of 0.5 mM ascorbate. Viability was assessed at 48 h post-treatment. Schematic diagrams summarize involvement of redox cycling between MnBuOE and a thiols (RS⁻) and b ascorbate (HA⁻). n.s, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001

Scheme 1

Major therapeutic effects, as well as the other aspects of development of Mn(III) meso-tetrakis(N-n-butoxyethylpyridinium-2-yl)porphyrin, MnTnBuOE-2-PyP⁵⁺ (MnBuOE, BMX-001), that guided its Clinical development [, –24]

Scheme 2

The proposed impact of MnP/H₂O₂-based redox system [TRAIL + CBX + MnBuOE + Ascorbate/Thiol(NAC)] on cellular metabolism