Phenylacetylglutaminate and Phenylacetate in Combination Upregulate VDUP1, Cause Cell Cycle Blockade and Apoptosis in U87 Glioblastoma Cells ()
1. Introduction
Phenylacetylglutaminate (PG) and Phenylacetate (PN) are metabolites of Phenylbutyrate (PB). Both PG and PN are naturally occurring in the human body as a result of metabolism of phenylalanine in the liver and kidneys [1]. The formulation of antineoplaston AS2-1 is a 4:1 mixture of synthetic PN and PG and A10 is a 4:1 mixture of PG and iso-PG. Antineoplastons A10 and AS2-1 have recently been granted FDA orphan drug designation for the treatment of all gliomas. Phase II trials indicate efficacy of antineoplastons in low-grade glioma, brain-stem glioma, high-grade glioma, including glioblastoma multiforme and primitive neuroectodermal tumors adenocarcinoma of the colon, and hepatocellular carcinoma [2,3]. Synthetic antineoplastons A10 and AS2-1 are now candidate drugs for Phase III trials [3]. Application of these two compounds in combination is novel and the molecular targets of PG have not been studied previously. Other studies have shown that PB and PN inhibit proliferation of human medulloblastoma cells [4]. PB is also indicated for the treatment of glioma and acute promyelocytic leukemia [4, 5]. PN has been examined independently in the past by other researchers as a potential anti-tumor agent in the treatment of prostate and thyroid cancer, glioma and hematological malignancies [5-9]. PN has been shown to cross the blood brain barrier [10]. PG on the other hand has been studied as a vehicle for nitrogen excretion in humans [11].
Studies using SKBR-3 breast cancer cells have shown that antineoplaston A10 downregulated expression of PKCa and inhibited cell proliferation due to arrest in G1 phase [12]. Antiproliferative activity of antineoplaston A10 and AS2-1 has also been reported in hepatocellular carcinoma, HepG2 and HLE cells [13,14].
Isobologram studies were introduced in the late 1920s [15,16] and are done to determine synergism, antagonism, or addition of effects of the drugs in combination [17]. An isobole is a graph of equally effective doses pairs for a specific effect level such as inhibitory concentration (IC50) [18].
Vitamin D upregulated protein (VDUP1) is a negative regulator of thioredoxin (Trx) and is also known as Thioredoxin interacting protein (TXNIP) [19]. Trx is an inhibitor of apoptosis and inhibits apoptosis signal regulating kinase-1 (ASK1) [20]. Increased levels of thioredoxin and reduced expression of VDUP1 has been found in many human cancers [19]. Up-regulation of VDUP1 has been shown to inhibit proliferation of tumor cells, identifying it as a novel tumor suppressor [20]. Overexpression of VDUP1 can occur due to various stresses such as serum starvation, H2O2, irradiation, heat shock as well as anti-cancer agents such as SAHA, anisomycin, and 5-fluorouracil [20,21].
It has been speculated that increased activation of DNA damage checkpoint pathways could play a role in the resistance of gliomas to chemoand radiation therapy [22,23]. Hence the search for chemotherapeutic agents that target such checkpoints has been ongoing. The G2/M checkpoint kinases Chk1 and Chk2 play an important role in tumorigenesis and are activated by kinases ATM and ATR [24]. Substrates of Chk1 and Chk2 include Cdc25A, Cdc25C and p53. Agents such as temozolomide cause G2/M arrest and activation of CHK1. This checkpoint activation prolongs the death of tumor cells and makes some cells resistant to treatment with temo-zolomide.
In this study we have attempted to identify the target pathways involved in the anti-proliferative/pro-apoptotic effect of PG and PN in combination. The results presented support the effectiveness of antineoplastons as anti-cancer therapy.
2. Materials and Methods
2.1. Preparation of PG and PN Solutions
Sodium salts of PG and PN were synthesized at Burzynski Research Institute. Stock solutions of these drugs were made in sodium-free MEM. In order to maintain the salt concentration in the medium at physiological levels, the salt from the drug was taken into account during media formulation.
2.2. Cell Culture
All human malignant cell lines were purchased from American Tissue Culture Collection (Rockville, MD). Cells were not used for more than 9 passages (3 months) post-resuscitation (Cell Line Verification Test Recommendations, ATCC Technical Bulletin No. 8 (2008)). U- 373 ATCC HTB-17, U87 ATCC-HTB-14, Daoy ATCC HTB-186, Bt-20 ATCC HTB-19 (F-12302), Hep G2 ATCC HB-8065. For all the experiments, U87 cells were maintained in complete modified Eagle’s medium (CMEM) at 37˚C, > 90% humidity, 5% CO2, for 3 days to allow cells to enter exponential growth phase. U87 cells were then trypsinized and re-plated at a density of 3000 cells/well in 96 well plates or 5000/cm2 in flasks based on each experiment for 3 days. After this period the culture medium was replaced with CMEM containing either 50 mM PG, 10 mM PN or a combination of 50 mMPG + 10mM PN. These concentrations were used in all the experiments.
2.3. Cell Proliferation Study
Cells were treated with PG, iso-PG or PN in concentrations indicated in the dose response curves for 3 days. The cells were washed with phosphate-buffered saline (PBS) and air-dried. Cellular protein was precipitated with 10% TCA in PBS for 1 hour at 4˚C. Plate was then rinsed 5 times with ddH2O and air dried for 2 hours. The dye sulforhodamine B (SRB) was applied at 0.4% for 30 minutes at room temperature. After an additional wash with 1% acetic acid, the plate was air-dried and unbuffered 10 mM Tris, pH 10.5 was added. Optical density was read at 540 nm. The data are shown as fractional survival (f) calculated by dividing the percent survival (optical density) in drug-treated wells by the percent survival (optical density) in the control wells.
2.4. Preparation of Drug Combinations in Fixed Ratios, Plotting of Isobole
A modified fixed ratio method was used to determine in vitro drug interactions in U87 cells [18]. PN and PG are used in combination in the formulation of antineoplaston AS2-1. In cell proliferation studies, we found that PN was 5 times more potent than PG. This potency ratio was used in combining both the drugs for isobologram studies. A series of dilutions of each drug were prepared so that the IC50 fell near the midpoint of the series. The highest concentrations of PG or PN were used to prepare fixed-ratio stock solutions at ratios of 5:0, 4:1, 3:2, 1:4 and 0:5. These solutions were then serially diluted in two-fold dilutions. Cells were treated for 3 days as described above. Cell proliferation was determined as above by SRB method. For each agent the ratio of 5:0 and 0:5 acted as single-agent (PG or PN) independent of the other and was used to plot dose response curves to obtain the individual IC50 by linear regression analysis each time the experiment was repeated. Solutions at ratios 4:1, 3:2, 2:3 and 1:4 gave dose response curves to find the IC50 of the drugs in combination. The IC50 of PG or PN was plotted on the horizontal and vertical axes and the line of additivity or isobole wasw drawn connecting the two IC50 levels. The experimental combinations of PG with PN were then analyzed in the same way to give the combination that caused isoeffect (IC50). This isoeffect value of the combination was plotted on the isobole. The combination of PG with PN was considered synergistic if the IC50 was plotted below; it was additive if plotted on, and antagonistic if plotted above the isobole.
2.5. Microarray Study
U87 cells were treated as before for 3 days with PG, PN or the combination. Total RNA was isolated from the cells in Trizol (Ambion) using standard protocols followed by RNeasy cleanup step (Qiagen). Biotin-labeled cRNA was synthesized from total RNA using standard procedures and was hybridized to the Affymetrix Human Genome U133 plus 2.0 arrays. Further quantitative data analysis to identify changes in gene expression patterns was done using GeneSpring v7.3 (Agilent). Briefly, the CEL files were normalized using MAS5 before being subjected to one way ANOVA (P < 0.05) and a Benjamini Hochberg False Discovery rate correction. The genes that were found to be statistically different were then filtered to create a list of genes differentially expressed at a 2- fold or greater level between controls and treated. These differentially expressed genes were subjected to functional cluster analysis using MAPPFinder in conjunction with GenMAPP (Gene Microarray Pathway Profiler) 2.0 and DAVID software.
2.6. Western Blot
After a 3 day treatment with PG, or PN, or the combination, floater cells were combined with the adherent cells that were collected by trypsinization. Cells were lysed in lysis buffer (20 mM Hepes pH 7.9, 100 mM KCl, 300 mM NaCl, 10 mM EDTA, 0.1% Nonidet P-40 plus protein inhibitor cocktail) for 1 - 3 hrs on ice and 20 ug of total protein was resolved on a 8% SDS-PAGE gel and transferred to nitrocellulose membrane. Blots were probed with either rabbit anti-VDUP1 0.1 ug/ml (Zymed laboratories, Invitrogen), anti-p21 0.5 ug/ml (Sigma), anti-p53 1 ug/ml (Sigma), or anti-GAPDH 0.5 ug/ml (Sigma) followed by secondary antibodies conjugated with alkaline phosphatase and developed with Pierce 1-step NBT/BCIP developing reagent. Band intensity was analyzed and quantified by GeneSnap software (Synoptics, Cambridge, UK).
2.7. Caspase Assay
Caspase 3 activity was determined using the colorimetric assay kit (Abcam). U87 cells were treated with PG, PN or the combination for 3 days after which floater and adherent cells were collected and counted. Equal number of cells were lysed in lysis buffer and pre-clarified by centrifugation (10000 × g) for 1min and used as per the kit to quantify Caspase 3 activity.
2.8. Measurement of DNA Synthesis by Thymidine Incorporation
U87 cells in log phase were plated in 96 well plates and the treatment carried out as previously described. The effect of each agent on DNA synthesis was estimated by the measurement of [methyl-3H] thymidine incorporation into TCA precipitable material after 2, 4, 24 and 36 hours. Thymidine was added at a concentration of 1 uCi/ml to the wells for a 2 h duration prior to each time point to avoid thymidine toxicity. Unincorporated [3H] thymidine, was removed by two washes with PBS supplemented with BSA (2 g/l). DNA was precipitated with 5% TCA (w/v) at 4˚C for 30 minutes. The precipitates were washed twice with 95% ethanol and dissolved in 1 ml of NaOH. Total counts were read in a liquid scintillation counter after neutralization with 1 ml of 1 M HCl. The incubations were performed in replicate and the mean values were used for the calculations.
2.9. Flow Cytometry/Apoptosis Assay
Apoptosis was measured using the Annexin V-FITC/ propidium iodide (PI) apoptosis kit (Sigma) according to the manufacturer’s instructions. Flow cytometry was used for detection of the membrane phospholipid, phosphotidylserine by Annexin V staining and for cell viability by PI exclusion. U87 cells were treated with 50 mM PG, 10 mM PN, 50 mM PG + 10 mM PN or cisplatin (Sigma) for 3 days. Adherent cells were trypsinized and pooled with floaters that were collected by centrifugation. The cells were resuspended in Annexin V binding buffer (150 mmol/L NaCl, 18 mmol/L CaCl2, 10 mmol/L HEPES, 5 mmol/L KCl, 1 mmol/L MgCl2). FITC conjugated Annexin V (1 ug/ml) and PI (50 ug/ml) were added to the cells and incubated for 30 minutes at room temperature. Analyses were performed on a FACScan (BD) and the data analyzed using CellQuest software (BD).
2.10. CHK1 Kinase Activity Assay
The K-lisa checkpoint activity kit (Calbiochem) was used as per instructions provided. Cells were treated with, 50 mM PG, 10 mM PN or a combination of 50 mM PG + 10 mM PN as described above. Cell lysate at 1 × 106/ml of lysis buffer was prepared. Briefly the assay is an ELISA based activity assay that uses a biotinylated peptide substrate that is phosphorylated on the fourth serine by CHK1. Biotinylated substrate and cell lysate from U87 cells were incubated in the presence of ATP in wells of Streptavidin-coated 96 well plate. Detection of the phosphorylated substrate is done using the phosphoserine detection antibody followed by HRP-antibody conjugate and color development with TMB substrate. Absorbance was read at dual wavelength 450/540 nm.
2.11. Flow Cytometry for Cell Cycle Study
U87 cells were treated with 50 mM PG, 10 mM PN or the combination of 50 mM PG + 10mM PN for each time point. Cells were trypsinized pelleted by centrifugation, resuspended in 1 ml ice-cold PBS with 2% serum and fixed by adding 4 ml of ice-cold ethanol under gentle vortexing. Fixed cells were collected by centrifugation, resuspended in 1 ml PBS and treated with 20 ug of DNasefree RNase (Sigma) for 30 min at 37˚C. Cells were stained with PI (100 ug/ml) for 10 minutes at room temperature. DNA content was analyzed on a FACScan. Raw data were gated to remove doublets and cellular debris. The cell cycle histograms were analyzed with CellQuest software.
2.12. Statistical Tests
All data were computed as the mean ±SD. The Student ttest was used to determine statistical significance at a criterion of P < 0.05.
3. Results
3.1. PG and PN Show Antiproliferative Effect in Different Cell Lines
All cell lines tested showed sensitivity to both PG and PN at different levels as shown in Figure 1. The IC50 for PG in the brain tumor cell lines was 50 mM for U87 and over 60 mM for U373 and DAOY. However the IC50 for the breast cancer line BT-20 was 55 mM and for HepG2 it
Figure 1. The cell lines, DAOY, U373, U87, BT-20 and Hep-G2 were treated with PG, iso-PG or PN at different concentrations for 3 days. Cell viability was determined by SRB dye. The bars mean ± SD of triplicate experiments compared to control untreated cells. The data is normalized and presented as a percent of control untreated cells.
was 45 mM PN on the other hand showed greater potency than PG with the IC50 falling between 9 mM and 11 mM, except in BT-20 where it was 14 mM. The data are shown as fractional survival, calculated by dividing the percent survival in drug-treated wells by the percent survival in the control well. For the remaining part of the paper we chose to use U87 cells as the model system.
3.2. PG and PN Act in a Synergistic or Additive Manner in Different Ratio Combinations
Isobologram analysis of PG and PN combinations show that the IC50 of the combinations lies below or on the line of additivity or isobole. This indicates that a combination of the two drugs is more effective than each drug used at the same concentration individually. In Figure 2 we have shown that the combination of PG and PN in the ratio 3:2 is additive as the IC50 falls on the isobole and the remaining combination at ratios 1:4, 2:3 and 4:1 are synergistic as the IC50 fall below the isobole.
3.3. Upregulation of VDUP1/TXNIP Protein in Cells Treated with PG and PN
Microarray analysis showed a remarkable up-regulation in the expression of VDUP1/TXNIP mRNA by 32 fold as compared to untreated cells as seen in Table 1. This was confirmed by qPCR (data not shown). Western blot analysis showed an increase in the level of VDUP1 protein in 48 and 72 hour exposure to PG and PN as seen in
Figure 2. Isobologram (solid line) was plotted using the IC50 of PG on the horizontal axis and the IC50 of PN on the vertical axis. The ratios for the combination were 4:1, 3:2, 2:3 and 1:4 to generate the dose response curves and the IC50 for the combinations. The 95% confidence intervals (dotted line) for the isobologram was calculated using the mean IC50 values from the single-agent dose response curves for PG or PN.
Figure 3. The expression of VDUP1 was significantly more enhanced by the treatment with the combination of PG and PN.