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Abstract

The purpose of this study was to elucidate the role of gamma-tocotrienol (GT3)-mobilized progenitors in mitigating damage to mice exposed to a supralethal dose of cobalt-60 gamma-radiation. CD2F1 mice were transfused 24 h post-irradiation with whole blood or isolated peripheral blood mononuclear cells (PBMC) from donors that had received GT3 72 h prior to blood collection and recipient mice were monitored for 30 days. To understand the role of GT3-induced granulocyte colony-stimulating factor (G-CSF) in mobilizing progenitors, donor mice were administered a neutralizing antibody specific to G-CSF or its isotype before blood collection. Bacterial translocation from gut to heart, spleen and liver of irradiated recipient mice was evaluated by bacterial culture on enriched and selective agar media. Endotoxin in serum samples also was measured. We also analyzed the colony-forming units in the spleens of irradiated mice. Our results demonstrate that whole blood or PBMC from GT3-administered mice mitigated radiation injury when administered 24 h post-irradiation. Furthermore, administration of a G-CSF antibody to GT3-injected mice abrogated the efficacy of blood or PBMC obtained from such donors. Additionally, GT3-mobilized PBMC inhibited the translocation of intestinal bacteria to the heart, spleen, and liver, and increased colony forming unit-spleen (CFU-S) numbers in irradiated mice. Our data suggests that GT3 induces G-CSF, which mobilizes progenitors and these progenitors mitigate radiation injury in recipient mice. This approach using mobilized progenitor cells from GT3-injected donors could be a potential treatment for humans exposed to high doses of radiation.

Drug preparation and administration

GT3 formulation in 5% Tween-80 in saline was purchased from Yasoo Health, Inc. (Johnson City, TN, USA). Olive oil was used as vehicle control (equivalent to the quantity of GT3) in 5% Tween-80. The final GT3 concentration was adjusted to administer a dose of 200 mg/kg in 0.1 ml. Control mice received 0.1 ml of vehicle. Mice were administered GT3 or vehicle subcutaneously (sc) at the nape of the neck with a 23 G needle attached to a Luer-Lock syringe 72 h before harvesting blood. No infections or local reactions were noted at the site of injection. AMD3100 (commercially known as plerixafor or Mozobil, Sigma-Aldrich, St. Louis, MO, USA) was diluted to 1.25 mg/ml with phosphate-buffered saline (PBS) for a dose of 5 mg/kg . It has been reported that AMD3100 aids in mobilizing progenitors from bone marrow to peripheral circulation and improves the yield of progenitors when injected to mice 1 h prior to harvest , . All donor mice, including those receiving the vehicle (olive oil), also received AMD3100 sc in a volume of 0.1 ml using a 23 G needle 1 h before blood collection to enhance mobilization .

2.1. Dose-Response of Atorvastatin and GT3 on TM Expression

To measure the effects of atorvastatin and GT3 on TM expression, primary human umbilical vein endothelial cells (HUVECs) were treated with different doses of atorvastatin and GT3 for 24 h and TM expression was measured by flow cytometry. We observed dose-dependent increases in TM expression after atorvastatin treatment (A). Treatment with 1, 2.5, and 5 μM of atorvastatin resulted in a 1.36-, 2.45-, and 4.46-fold increase in TM expression, respectively, as compared to the vehicle treated group. GT3 treatment at 2.5 μM did not cause a significant change in TM expression, but 5 μM GT3 treatment for 24 h caused a 2.35-fold increase in TM expression (B).

2.2. Atorvastatin and GT3 Synergistically Up-Regulate TM Expression

Next, we investigated whether atorvastatin and GT3 has a synergistic effect on TM expression. To address the issue, we used low doses of atorvastatin and GT3. HUVECs were treated with 1 μM of atorvastatin, 1 μM of GT3, and 1 μM of atorvastatin plus GT3. They were also treated with 2.5 μM of atorvastatin, 2.5 μM of GT3, and 2.5 μM of atorvastatin plus 2.5 μM of GT3 for 24 h, and TM expression was measured in all groups by flow cytometry. For both the treatment strategies, we found that combined treatment resulted in significantly (p < 0.01) higher TM expression than singular treatment with atorvastatin or GT3 (A,B and ). TM expression was unaltered after 1 μM and 2.5 μM of GT3 treatment, but it was increased 1.36- and 2.45-fold after 1 μM and 2.5 μM, respectively, of atorvastatin treatment. Interestingly, TM expression increased 1.6-fold after treatment with 1 μM of atorvastatin plus 1 μM GT3, and it increased 3.38-fold after treatment with 2.5 μM atorvastatin plus 2.5 μM GT3. These findings show that atorvastatin and GT3 synergistically induce TM expression in this used dose range. However, when we treated HUVECs with 5 μM atorvastatin plus 5 μM GT3, the TM expression reduced to control level at 24 h ().

2.3. Atorvastatin and GT3 Synergistically Induce TM Functional Activity

Next, we determined whether atorvastatin and GT3 synergistically increased the functional activities of TM, which was measured by APC generation assay. HUVECs were treated with atorvastatin alone, GT3 alone, and atorvastatin plus GT3 for 24 h. We used three different doses of GT3 (1, 2.5, and 5 μM) and two doses of atorvastatin (1 and 2.5 μM). No significant change in APC generation was observed after the 1, 2.5 and 5 μM GT3 treatments. However, the 1 and 2.5 μM atorvastatin treatment for 24 h significantly (p < 0.001) induced APC generation when curve slopes were compared between vehicle-treated and atorvastatin-treated groups (). APC generation was maximally induced after combined treatment with 1 μM atorvastatin plus 1 μM GT3, 1 μM atorvastatin plus 2.5 μM GT3, 1 μM atorvastatin plus 5 μM GT3, and 2.5 μM atorvastatin plus 2.5 μM GT3 treatment compared to treatment with each drug alone (A–D), suggesting that atorvastatin and GT3 synergistically enhance the functional activity of TM as well as TM expression in this dose range.

2.4. Atorvastatin and GT3 Synergistically Up-Regulate KLF2 Expression

Since the transcription factor Kruppel-like factor 2 (KLF2) plays an important role in modulating TM expression, we hypothesized that atorvastatin and GT3 would also synergistically up-regulate KLF2 expression. HUVECs were treated with 2.5 μM of atorvastatin, 2.5 μM of GT3, and 2.5 μM of atorvastatin plus 2.5 μM of GT3. KLF2 mRNA expression was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) at three different time intervals (6, 12, and 24 h). GT3 treatment alone was not able to induce KLF2 expression at the 6 and 12 h time points; however, there was an approximately 2.5-fold increase (p = 0.0006) at the 24 h time point (A–C). On the other hand, treatment with atorvastatin alone significantly induced (p < 0.001) KLF2 expression at all three time points (A–C), but a maximum increase in KLF2 expression was observed after combined treatment with atorvastatin and GT3, suggesting a synergistic effect of these two compounds on KLF2 expression (A–C).

Abstract

The development of radiation countermeasures for acute radiation syndrome (ARS) has been underway for the past six decades, leading to the identification of multiple classes of radiation countermeasures. However, to date, only two growth factors (Neupogen and Neulasta) have been approved by the United States Food and Drug Administration (US FDA) for the mitigation of hematopoietic acute radiation syndrome (H-ARS). No radioprotector for ARS has been approved by the FDA yet. Gamma-tocotrienol (GT3) has been demonstrated to have radioprotective efficacy in murine as well as nonhuman primate (NHP) models. Currently, GT3 is under advanced development as a radioprotector that can be administered prior to radiation exposure. We are studying this agent for its safety profile and efficacy using the NHP model. In this study, we analyzed global metabolomic and lipidomic changes using ultra-performance liquid chromatography (UPLC) quadrupole time-of-flight mass spectrometry (QTOF-MS) in serum samples of NHPs administered GT3. Our study, using 12 NHPs, demonstrates that alterations in metabolites manifest only 24 h after GT3 administration. Furthermore, metabolic changes are associated with transient increase in the bioavailability of antioxidants, including lactic acid and cholic acid and anti-inflammatory metabolites 3 deoxyvitamin D3, and docosahexaenoic acid. Taken together, our results show that the administration of GT3 to NHPs causes metabolic shifts that would provide an overall advantage to combat radiation injury. This initial assessment also highlights the utility of metabolomics and lipidomics to determine the underlying physiological mechanisms involved in the radioprotective efficacy of GT3.

1. Introduction

Here, we have studied GT3 induced changes in metabolomic and lipidomic profiles as part of its safety and toxicity testing in the NHP model. We report GT3-induced longitudinal changes in metabolic and lipidomic serum profiles of NHPs using a global metabolomic and lipidomic approach with an ultra-performance liquid chromatography (UPLC) quadrupole time-of-flight mass spectrometry (QTOF-MS) platform. We found few changes in metabolite profiles during the first 24 h following GT3 administration. There were significant changes in profiles of several metabolites over time (1–3 days) that returned to baseline levels over the next 20-day follow-up period. Furthermore, there was a significant overlap of metabolite profiles for the three doses of GT3 that were tested in this study. The findings from metabolic profiles were consistent with complete blood count (CBC) and blood chemistry analyses of the same set of animals post GT3 administration. Taken together, these results suggest that GT3 administration does not have adverse consequences on overall metabolism and is safe for use as a potential radiation countermeasure.

2.1. Identification of Metabolite Signatures of GT3 Administration in NHPs

Untargeted metabolomic and lipidomic profiling was performed for a total of 208 serum samples obtained from 12 NHPs (four per dosage group) in this pilot study. All NHPs were included in the analysis since there were no outliers. A total of 3667 features in the Electrospray Ionization (ESI) positive mode and 4914 features in the ESI negative mode were detected for metabolomics (Acquity BEH C18 column) while a similar analysis of lipidomics (Acquity CSH C18 column) yielded 1573 features in the ESI positive and 622 in ESI negative mode. Scores plots () were analyzed to evaluate group separation resulting from inherent differences in metabolite profiles of NHPs that received 9.375, 18.75, and 37.5 mg/kg of GT3 as compared to the pre-administration samples. Partial least squares-discriminant analysis (PLS-DA) plots of the negative ionization compounds and lipids are presented in . The coefficient of variation (CV) values of internal standards for quality control samples (QCS) as well as the total ion chromatogram (TIC) overlays have been presented as . We asked if GT3 administration induced changes in profiles in the short-term (first 24 h) (a) and/or if overall metabolite profiles changed over the long-term (2 days to 20 days) (b). Each time point following GT3 administration was defined as an independent class for multivariate analysis. Principal component analysis did not yield separation for any of the models. PLS-DA multi-class models including two components yielded R2 = 0.52 and Q2 = 0.19 for the short-term group and R2 = 0.43 and Q2 = 0.07 for the long-term group. There was a significant overlap between the treated groups with the pre-injection samples for time points monitored within the first 24 h, demonstrating that GT3 administration induced minimal changes in overall metabolite profiles. There was a modest separation of treated groups for later time points (1–3 days). Additionally, there was a significant overlap between groups for different doses of GT3. This suggests that a sample size of four may not be large enough to observe dose-dependent metabolic changes when the difference in GT3 concentration is only two-fold. Thus, we determined the mean for results obtained with three different doses of GT3 at each time point to compare with pre-drug injection samples.

2.1. Identification of Metabolite Signatures of GT3 Administration in NHPs

Next, chemical formula calculations for 2323 significantly dysregulated metabolites (using ANOVA, adjusted p < 0.05) were performed via accurate mass based search and the identity of 61 metabolites was confirmed using tandem mass spectrometry (). We used hierarchical clustering to visualize time and/or dose dependence of these validated metabolites, as a heat map (). While there were no appreciable changes in relative abundance within 24 h, we observed transient changes in abundance 1–3 days post-administration of GT3, and the endogenous levels returned to normal over the extended time course.

Abstract

Natural antioxidant gamma-tocotrienol (GT3), a vitamin E family member, provides intestinal radiation protection. We seek to understand whether this protection is mediated via mucosal epithelial stem cells or sub-mucosal mesenchymal immune cells. Vehicle- or GT3-treated male CD2F1 mice were exposed to total body irradiation (TBI). Cell death was determined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Villus height and crypt depth were measured with computer-assisted software in tissue sections. Functional activity was determined with an intestinal permeability assay. Immune cell recovery was measured with immunohistochemistry and Western blot, and the regeneration of intestinal crypts was assessed with ex vivo organoid culture. A single dose of GT3 (200 mg/kg body weight (bwt)) administered 24 h before TBI suppressed cell death, prevented a decrease in villus height, increased crypt depth, attenuated intestinal permeability, and upregulated occludin level in the intestine compared to the vehicle treated group. GT3 accelerated mesenchymal immune cell recovery after irradiation, but it did not promote ex vivo organoid formation and failed to enhance the expression of stem cell markers. Finally, GT3 significantly upregulated protein kinase B or AKT phosphorylation after TBI. Pretreatment with GT3 attenuates TBI-induced structural and functional damage to the intestine, potentially by facilitating intestinal immune cell recovery. Thus, GT3 could be used as an intestinal radioprotector.

2.1. Experimental Model, GT3 Treatment, Irradiation, Tissue Procurement, and Euthanasia

All animal studies were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal protocol (AUP #3823) was approved by the Institutional Animal Care and Use Committee of the University of Arkansas for Medical Sciences (UAMS). Male CD2F1 mice were obtained from Charles River Laboratories (Houston, TX); mice between 8 and 12 weeks of age were used for experiments. They were housed in conventional cages in a pathogen-free environment with controlled humidity, temperature, and a 12–12 light–dark cycle with free access to drinking water and standard chow (Teklad, Madison, WI, USA).

2.1. Experimental Model, GT3 Treatment, Irradiation, Tissue Procurement, and Euthanasia

GT3 was prepared freshly before administration with 5% Tween-80 (Fisher Scientific, Waltham, MA, USA) in saline. A single dose of 100 µL vehicle or GT3 (200 mg/kg body weight (bwt)) was administered subcutaneously 24 h before TBI.

Abstract

Tocotrienol, an analogue of vitamin E has been known for its numerous health benefits and anti-cancer effects. Of the four isoforms of tocotrienols, gamma-tocotrienol (γT3) has been frequently reported for their superior anti-tumorigenic activity in both in vitro and in vivo studies, when compared to its counterparts. In this study, the effect of γT3 treatment in the cytoplasmic and nuclear fraction of MDA-MB-231 human breast cancer cells were assessed using the label-free quantitative proteomics analysis. The cytoplasmic proteome results revealed the ability of γT3 to inhibit a group of proteasome proteins such as PSMA, PSMB, PSMD, and PSME. The inhibition of proteasome proteins is known to induce apoptosis in cancer cells. As such, the findings from this study suggest γT3 as a potential proteasome inhibitor that can overcome deficiencies in growth-inhibitory or pro-apoptotic molecules in breast cancer cells. The nuclear proteome results revealed the involvement of important nuclear protein complexes which hardwire the anti-tumorigenesis mechanism in breast cancer following γT3 treatment. In conclusion, this study uncovered the advancing roles of γT3 as potential proteasomes inhibitor that can be used for the treatment of breast cancer.

2.1. Cell Line and Treatment Conditions

The MDA-MB-231 cells were grown in T75 flasks in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) in a humidified atmosphere of 5% carbon dioxide in air at 37 °C, until it reaches 70–90% confluency. A total of 5 × 105 cells were seeded in T75 flask containing 10 mL of complete media. Following cell adherence, the cells were treated with IC50 concentration, 5.8 µg × mL−1 of γT3, as established previously []. Following 24 h of γT3 treatment, the cells were washed with 1× PBS solution. A cell scrapper was used to dislodge the attached cells. The cells were collected in 5 mL PBS and washed with two volumes of ice-cold PBS and centrifuged at 250 × g for 5 min at 4 °C. The supernatant was discarded, and the pellets were resuspended in 40 mL of ice-cold PBS and centrifuged as before. The washing step was repeated twice. The pellet was subjected to two separate processing steps to extract (i) cytoplasmic proteins and (ii) nuclear proteins.

3.1. Effect of Beta- and Gamma- Tocotrienols on the Cell Proliferation of MDA-MB-231 and MCF7 cells

Using WST-1 as a cell proliferation reagent, the percent proliferation of the MDA-MB-231 cell line treated with different concentrations of beta-T3 (10–50 µM) or gamma-T3 (10–50 µM) for 24 and 48 h was calculated and the results showed a significant dose- and time-dependent decrease in the proliferation of both cell lines; however, the effect was more prominent with beta-T3 treatment. Beta-tocotrienol induced a significant progressive decrease in percentage of proliferating MDA cells, with an IC50 of 29.99 μM and 21.14 μM after 24 and 48 h respectively (A). On the other hand, gamma-tocotrienol induced a significant progressive decrease in cell proliferation of MDA cells starting from 30 μM with an IC50 of 39.04 μM and 30.98 μM after 24 and 48 h respectively (B). The IC50 concentrations of beta-T3 were lower than that of the gamma derivative after both 24 and 48 h treatments, indicating a significant higher potency of beta-T3 on MDA cells at 20, 30 and 40 μM (C,D).