High levels of ubidecarenone (oxidized CoQ10) delivered using a drug-lipid conjugate nanodispersion (BPM31510) differentially affect redox status and growth in malignant glioma versus non-tumor cells

Abstract

Metabolic reprogramming in cancer cells, vs. non-cancer cells, elevates levels of reactive oxygen species (ROS) leading to higher oxidative stress. The elevated ROS levels suggest a vulnerability to excess prooxidant loads leading to selective cell death, a therapeutically exploitable difference. Co-enzyme Q₁₀ (CoQ₁₀) an endogenous mitochondrial resident molecule, plays an important role in mitochondrial redox homeostasis, membrane integrity, and energy production. BPM31510 is a lipid-drug conjugate nanodispersion specifically formulated for delivery of supraphysiological concentrations of ubidecarenone (oxidized CoQ₁₀) to the cell and mitochondria, in both in vitro and in vivo model systems. In this study, we sought to investigate the therapeutic potential of ubidecarenone in the highly treatment-refractory glioblastoma. Rodent (C6) and human (U251) glioma cell lines, and non-tumor human astrocytes (HA) and rodent NIH3T3 fibroblast cell lines were utilized for experiments. Tumor cell lines exhibited a marked increase in sensitivity to ubidecarenone vs. non-tumor cell lines. Further, elevated mitochondrial superoxide production was noted in tumor cells vs. non-tumor cells hours before any changes in proliferation or the cell cycle could be detected. In vitro co-culture experiments show ubidecarenone differentially affecting tumor cells vs. non-tumor cells, resulting in an equilibrated culture. In vivo activity in a highly aggressive orthotopic C6 glioma model demonstrated a greater than 25% long-term survival rate. Based on these findings we conclude that high levels of ubidecarenone delivered using BPM31510 provide an effective therapeutic modality targeting cancer-specific modulation of redox mechanisms for anti-cancer effects.

Figure 1

Differential effects of oxidized CoQ₁₀ on glioblastoma and non-tumor cell lines. (A) Relative cell viability of rodent C6 glioma and human U251 glioma cells after exposure to 0.1% DMF, 10 µM native CoQ10 (in 0.1% DMF), or 10 µM of ubidecarenone using BPM31510 for 72 h. Values are normalized to control. No significant effect on cell growth is noted under any condition. (B) Dose response curves for rat C6 glioma and mouse NIH3T3 fibroblast cells (left panel) and human U251 glioma and HA cells (right panel) after incubation for 72 h with BPM31510. Note that in each case, the tumor line was more sensitive than the control. (C) Cell viability analysis of each cell line over time after incubation with increasing doses of ubidecarenone. The number of live cells is converted from a pre-established standard curve with known cells numbers. All data presented as Mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to control (no BPM31510) counts on the same day. Each color corresponds to the BPM31510 dose.

Figure 2

Ubidecarenone induces G₂/M cell-cycle arrest of glioblastoma but not non-cancerous cell lines. (A) Cell cycle analysis and quantification of human U251 glioma cells treated with 0, 75, 115, 230, 345, or 460 µM ubidecarenone. The percentage of cells in each phase (G_0/1, S or G₂ + M) is estimated from the frequency histograms. The percentage of cells in G₂ + M phase is highlighted. (B) Quantification of human U251 glioma, rat C6 glioma, HA, or mouse NIH3T3 fibroblast cells in each cell phase. All data presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to control (no drug exposure) at the same cell cycle phase.

Figure 3

Differential redox vulnerabilities to ubidecarenone exposure between non-tumor and glioblastoma cells. (A) Flow cytometry analysis of O₂⁻ in HA or human U251 glioma cells treated with 0, 115, or 460 µM ubidecarenone. O₂^- intensity in each cell is demonstrated in the frequency histograms. (B) Quantification of mean O₂⁻ levels in HA and human U251 glioma cells. A modest increase in O₂⁻ is noted in HA in the presence of ubidecarenone, although this increase is not dose-dependent. In contrast, there is a fourfold O₂⁻ increase in rat C6 glioma cells in a dose-dependent manner. (C) Flow cytometry analysis (scatter plot) of O₂⁻ and DAPI in human U251 glioma and HA cells. Notably, there is minimal change in O₂⁻ intensity with increasing ubidecarenone dose in HA cells, while there is a marked increase in both O₂⁻ and DAPI in human U251 glioma cells after drug exposure. (D) Quantification of relative cell populations of O₂⁻_low/DAPI_low, O₂⁻_high/DAPI_low, and O₂⁻_high/DAPI_high in human U251 glioma cells demonstrating increasing numbers of high O₂^- and DAPI labeled cells with increasing ubidecarenone dose. (E) Flow cytometry analysis of O₂⁻ for human U251 glioma cells treated with 230 µM ubidecarenone for 0, 2, 6, or 24 h. Relative mean values of O₂⁻ were quantified and normalized to control at 0 h. (F) Cell cycle analysis by flow cytometry for human U251 glioma cells treated with 230 µM ubidecarenone for 0, 2, 6, or 24 h. The percentage of human U251 glioma cells in each cell cycle phase (G_0/1, S or G₂ + M) was quantified. All data presented as Mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant, compared to control (0 µM) at the same cell cycle phase.

Figure 4

Ubidecarenone induces differential effects on cell growth and redox vulnerabilities between non-tumor and glioblastoma cells in co-culture. (A) Phase and fluorescent images of GFP-labeled rat C6 glioma cells and non-labeled mouse NIH3T3 fibroblast, co-cultured and treated with 0, 230, or 460 µM ubidecarenone, demonstrates a dose-dependent decrease in glioma cells with a relative sparing of HA cells. (B) Flow cytometry analysis (scatter plot) of GFP and O₂⁻ in GFP-labeled rat C6 glioma cells and non-labeled mouse NIH3T3 co-culture. Cell populations are characterized based on GFP intensity. Note the increase in the GFP_neg population relative to GFP_high population in the presence of ubidecarenone. (C) Flow cytometry analysis of GFP-labeled rat C6 glioma cells and non-labeled mouse NIH3T3 fibroblasts co-cultures treated with ubidecarenone for up to 12 days. Cells are characterized based on their GFP intensity (GFP_neg or GFP_pos). Results are grouped based on ubidecarenone dose and are shown as a percentage of the entire cell population. While glioma cells (GFP_pos) represent the entire cell population by day 9 in co-cultures without ubidecarenone, there are essentially equal numbers of glioma and non-tumor cell populations at doses ≥ 115 µM ubidecarenone.

Figure 5

Ubidecarenone induces differential effects on cell growth and redox vulnerabilities between non-tumor and glioblastoma cells in co-culture experiments. (A) Phase and fluorescent images of GFP-labeled human U251 glioma cells and non-labeled HA cells co-cultured and treated with 0, 230, or 460 µM ubidecarenone demonstrate a dose-dependent decrease in glioma cells with a relative sparing of HA. (B) Flow cytometry analysis (scatter plot) of GFP and O₂^- in GFP-labeled human U251 glioma cells and non-labeled HA cells in co-culture. Cell populations are characterized based on GFP intensity (GFP_neg, GFP_low, and GFP_high). Note the dose-dependent increase in the GFP_neg population relative to the GFP_high population in the presence of ubidecarenone. Quantification of flow cytometry cell populations illustrates a dose-dependent increase in GFP_low cell numbers. (C) Flow cytometry analysis of O₂⁻ and DAPI in GFP-labeled human U251 glioma cells and non-labeled HA cells, co-cultured and treated with 0, 230, or 460 µM ubidecarenone. Note the increase in O₂⁻ values for both GFP_low and GFP_high cells, with insignificant changes noted in the GFP_neg (HA cells) population. (D) Graphical depiction of flow cytometry analysis of O₂⁻ and DAPI in GFP-labeled human U251 glioma cells and non-labeled HA cells, co-cultured and treated with 0, 230, or 460 µM ubidecarenone. In contrast to the GFP_neg population, BPM exposure results in a marked increase in superoxide production in both the low and high GFP fractions. (E) DAPI levels are significantly elevated only in the GFP_low cell population, consistent with this population representing dying cells.

Figure 6

Ubidecarenone demonstrates efficacy in an orthotopic glioma model. Wistar rats received either saline or 50 mg/kg BPM31510 IP, twice daily, five days per week, starting at either 4 (n = 12) or 8 (n = 19) days after implantation of 10⁶ C6 glioma cells into the right striatum. (A) Survival of rats treated either with saline (n = 32) or BPM31510. Over 25% of BPM31510 treated rats survived to the end of experiment (at least 75 days) compared to 0% of PBS treated controls (P < 0.01, log rank statistic). No significant difference was noted between the BPM31510 treated groups starting at 4- or 8-days. (B) Serial MRI of a long-term survivor (Day 27, Day 34, and Day 102 post-implantation) demonstrating persistent effects even after treatment was withdrawn. Lower right panel is a coronal plane H&E stained section of the same long-term survivor demonstrating a cystic cavity with no obvious tumor present.