Metabolomics profiles delineate uridine deficiency contributes to mitochondria-mediated apoptosis induced by celastrol in human acute promyelocytic leukemia cells

Celastrol, extracted from “Thunder of God Vine”, is a promising anti-cancer natural product. However, its effect on acute promyelocytic leukemia (APL) and underlying molecular mechanism are poorly understood. The purpose of this study was to explore its effect on APL and underlying mechanism based on metabolomics. Firstly, multiple assays indicated that celastrol could induce apoptosis of APL cells via p53-activated mitochondrial pathway. Secondly, unbiased metabolomics revealed that uridine was the most notable changed metabolite. Further study verified that uridine could reverse the apoptosis induced by celastrol. The decreased uridine was caused by suppressing the expression of gene encoding Dihydroorotate dehydrogenase, whose inhibitor could also induce apoptosis of APL cells. At last, mouse model confirmed that celastrol inhibited tumor growth through enhanced apoptosis. Celastrol could also decrease uridine and DHODH protein level in tumor tissues. Our in vivo study also indicated that celastrol had no systemic toxicity at pharmacological dose (2 mg/kg, i.p., 21 days). Altogether, our metabolomics study firstly reveals that uridine deficiency contributes to mitochondrial apoptosis induced by celastrol in APL cells. Celastrol shows great potential for the treatment of APL.


Metabolomics profiles delineate uridine deficiency contributes to mitochondria-mediated apoptosis induced by celastrol in human acute promyelocytic leukemia cells EXTENDED EXPERIMENTAL PROCEDURES Cell proliferation assay
Briefly, after being seeded in a 96-well microplate at a density of 2×10 5 /well, the HL-60 cells were treated with increasing concentrations of celastrol (0.125, 0.25, 0.5, 1, 2, 4 and 8 μM) for 24 and 48 h. Then 10 μl CCK-8 solution was added to each well. After incubation at 37°C for an additional 4 h , the absorbance was measured at 450 nm with the microplate reader (Tecan Infinite 200, Switzerland).

Transmission electron microscope analysis
After celastrol treatment, the HL-60 cells were washed with ice-cold PBS for two times. Then the cells were fixed by 2.5% glutaraldehyde solution overnight at 4°C. The next day the cells were post-fixed with 1% osmium tetroxide. After being dehydrated in increasing concentrations of alcohol, the cell pellets were embedded in epon. Once ultrathin sections were cut, the cells were stained with uranyl acetate and lead citrate. At last, the ultrastructure of the cells was analyzed by transmission electron microscope.

RNA isolation and real-time PCR
Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The concentration of total RNA was determined using a NanoDrop 2000 (Thermo Fisher Scientific, Wilmington, DE). Reverse transcription and real-time PCR were performed according to the manufacturer's instructions (Takara, Tokyo, Japan). ACTB was used as an endogenous control to determine the expression of target genes by relative quantitation method. Quantification was carried out using ABI7900 Fast Real-Time System (Applied Biosystems, CA, USA) according to the manufacturer's instructions. The specificity of The PCR products was confirmed using melting curve analyses. The 2 −ΔΔCt method was used to calculate the relative expression of the target genes. All real-time PCR experiments were repeated at least three times.
Samples containing about 50 μg of total cellular protein were separated via electrophoresis through a SDS-polyacrylamide gel followed by transfer to a PVDF membrane (Millipore, Billerica, MA). Membranes were blocked for 1 h with 5% (w/v) nonfat milk in TBST at room temperature. The blots were incubated with primary rabbit polyclonal antibodies against cleaved caspase 9, cleaved caspase 3, Bax, p53 (Cell Signaling Technology, Beverly, MA, USA), rabbit monoclonal antibody against DHODH(Abcam, Cambridge, UK) and mouse monoclonal antibody against β-actin (Beyotime, China) at 4°C overnight. After being washed with TBST for three times, the membranes were incubated with HRP-conjugated secondary antibody (Beyotime, China) for 1 h at room temperature. The levels of target proteins were detected by ChemiDoc™ MP imaging system (Bio-Rad). Bands were monitored using Immobilon™ Western Chemiluminescent HRP Substrate (Millipore, USA). Western blots were quantified by the Image-Pro Plus software.

Metabolomics analysis
Metabolic profiling was performed on a UPLC Ultimate 3000 system (Dionex, Germering, Germany), coupled to an Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) in both positive and negative mode simultaneously. The chromatographic separation was performed on a 1.9 μm Hypersile Gold C18 column (100 mm×2.1 mm) (Thermo Fisher Scientific), and the column was maintained at 40°C. A multistep gradient had mobile phase A of 0.1% formic acid in ultrapure water and mobile phase B consisting of acetonitrile (ACN) acidified with 0.1% formic acid. The gradient operated at a flow rate of 0.4 ml/min over a run time of 15 min. The UPLC autosampler temperature was set at 4°C and the injection volume for each sample was 10 μl. All samples were analyzed in a randomized fashion to avoid complications related to the injection order. MS data were collected by the Orbitrap mass spectrometer equipped with a heated electrospray source (HESI). For both positive and negative mode, the operating parameters were as follows: a spray voltage of 3.5 kV for positive, 2.5 kV for negative, the capillary temperature of 300°C, sheath gas flow of 50 arbitrary units, auxiliary gas flow of 13 arbitrary units, sweep gas of 0 arbitrary units and S-Lens RF level of 60. In the full scan analysis (70 to 1050 amu), the resolution was set at 700,000 with an automatic gain control (AGC) target of 3×10 6 charges. The MS system was calibrated according to the manufacturer's instructions. The chemical identification is based on the retention time and accurate mass with commercial standards. Among the Qc samples, most of the metabolites showed RSDs less than 30%, indicating the metabolomics profiling was reliable [1].

Targeted uridine analysis in xenograft tumor in BALB/c nude mice
50 mg homogenized tumor tissue were mixed with 150 μl ultra-pure water and 600 μl pure methanol.
The tissues were ultrasonicated for 5 min (power: 60%, pulses: 3/3), and the supernatant was obtained after centrifugation at 20,000×g for 15 min. After dryness, the residue was reconstituted and then injected into a UPLC Ultimate 3000 system (Dionex), coupled to a Q-Exactive mass spectrometer (Thermo Fisher Scientific) for uridine analysis. The conditions of the mass spectrometer and chromatograph were the same as described in metabolomics analysis. The [M-H]ions of uridine at m/z 243.06226 were monitored.