table_1_Inhibiting Glycine Decarboxylase Suppresses Pyruvate-to-Lactate Metabolism in Lung Cancer Cells.docx

<p>Glycine decarboxylase (GLDC) gene is frequently upregulated in various types of cancer including lung, prostate and brain. It catabolizes glycine to yield 5,10-methylenetetrahydrofolate, an important substrate in one-carbon metabolism for nucleotide synthesis. In this study, we used exon splicing modulating steric hindrance antisense oligonucleotide (shAON) to suppress GLDC expression and investigated its effect on pyruvate metabolism via hyperpolarized carbon-13 magnetic resonance spectroscopy (MRS). The MRS technique allows us to study in vivo metabolic flux in tumor tissues with/without GLDC-shAON intervention. Here, we show that GLDC-shAON treatment is able to suppress lung cancer cell growth and tumorigenesis, both in vitro and in vivo. The carbon-13 MRS results indicated that the conversion of pyruvate into lactate in GLDC-shAON-treated tumor tissues was significantly reduced, when compared with the control groups. This observation corroborated with the reduced activity of lactate dehydrogenase and pyruvate dehydrogenase in GLDC-shAON-treated lung cancer cells and tumor tissues. Glycolysis stress test showed that extracellular acidification rate was significantly suppressed after GLDC-shAON treatment. Besides lung cancer, the antitumor effect of GLDC-shAON was also observed in brain, liver, cervical, and prostate cancer cell lines. Furthermore, it enhanced the treatment efficacy of cisplatin in lung cancer cells. Taken together, our findings illustrate that pyruvate metabolism decreases upon GLDC inhibition, thereby starving cancer cells from critical metabolic fuels.</p

image_1_Inhibiting Glycine Decarboxylase Suppresses Pyruvate-to-Lactate Metabolism in Lung Cancer Cells.tif

<p>Glycine decarboxylase (GLDC) gene is frequently upregulated in various types of cancer including lung, prostate and brain. It catabolizes glycine to yield 5,10-methylenetetrahydrofolate, an important substrate in one-carbon metabolism for nucleotide synthesis. In this study, we used exon splicing modulating steric hindrance antisense oligonucleotide (shAON) to suppress GLDC expression and investigated its effect on pyruvate metabolism via hyperpolarized carbon-13 magnetic resonance spectroscopy (MRS). The MRS technique allows us to study in vivo metabolic flux in tumor tissues with/without GLDC-shAON intervention. Here, we show that GLDC-shAON treatment is able to suppress lung cancer cell growth and tumorigenesis, both in vitro and in vivo. The carbon-13 MRS results indicated that the conversion of pyruvate into lactate in GLDC-shAON-treated tumor tissues was significantly reduced, when compared with the control groups. This observation corroborated with the reduced activity of lactate dehydrogenase and pyruvate dehydrogenase in GLDC-shAON-treated lung cancer cells and tumor tissues. Glycolysis stress test showed that extracellular acidification rate was significantly suppressed after GLDC-shAON treatment. Besides lung cancer, the antitumor effect of GLDC-shAON was also observed in brain, liver, cervical, and prostate cancer cell lines. Furthermore, it enhanced the treatment efficacy of cisplatin in lung cancer cells. Taken together, our findings illustrate that pyruvate metabolism decreases upon GLDC inhibition, thereby starving cancer cells from critical metabolic fuels.</p

Effect of ACS84 on oxidative stress induced by 6-OHDA in SH-SY5Y cells.

<p>(A) Dose dependent effect of ACS84 on ROS generation in the 6-OHDA-treated (50 µM) SH-SY5Y cells. Cells were pretreated with ACS84 at different concentrations for 4 h. DCFDAH₂ (10 µM) was given 30 min before the addition of 6-OHDA (50 µM). The results were obtained after the treatment with 6-OHDA for 1 h. (B–C) Effect of ACS84, L-Dopa and NaHS at 10 µM on ROS generation (B) and SOD (C) in SH-SY5Y cells treated with 6-OHDA. SOD activity was measured 4 h after 6-OHDA treatment. Data are presented as mean ± SEM, n = 4–8, ^###<i>P</i><0.001 versus control; *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, versus 6-OHDA-treated cells; ^†††<i>P</i><0.001, versus ACS84-treated cells.</p

Effect of ACS84 on 6-OHDA-induced TH+ neuronal degeneration.

<p>Immunofluorescence staining showed that ACS84 (10 mg kg⁻¹ day⁻¹, i.g) alleviated TH+ neuron loss in SN of 6-OHDA-lesioned PD rats. Photos were taken at x100 magnification, and the white bar indicated 0.1 µm. Samples were collected from two independent experiments.</p

Effect of ACS84 on oxidative stress in the striatum of unilateral 6-OHDA-lesioned PD rat model.

<p>ACS84 treatment (10 mg kg⁻¹ day⁻¹, i.g) alleviated the increased MDA production. Data are presented as mean ± SEM, n = 4–6. *<i>P</i><0.05 versus lesion site of Sham group and ^#<i>P</i><0.05 versus lesion site of Vehicle group.</p

Effects of ACS84 on the expression and translocation of antioxidant enzymes in SH-SY5Y cells.

<p>(A) Western blotting analysis showing that treatment with ACS84 for 4 h promoted the nuclear accumulation of Nrf-2 in SH-SY5Y cells. Densitometric analysis performed by normalizing nuclear Nrf-2 to cytosol Nrf-2 signals. Data were expressed as mean ± SEM, *<i>P</i><0.05, n = 5 (B) RT-PCR showing that ACS84 treatment induced the mRNA expression of GclC, GclM and HO-1 after 4 h. Samples were collected from three independent experiments.</p

Protective effect of ACS84 against cell injury induced by 6-OHDA in SH-SY5Y cells.

<p>(A–B): Dose dependent effects of ACS84 on (A) cell viability and (B) LDH release in the 6-OHDA-treated (50 µM) SH-SY5Y cells. Cells were pretreated with ACS84 at different concentrations for 1 h before the addition of 6-OHDA. The results were obtained at 12 h (MTT assay) or 6 h (LDH release assay) after the treatment with 6-OHDA. (C–D): Effect of ACS84, L-Dopa and NaHS at 10 µM on cell viability (C) and LDH release (D) in SH-SY5Y cells treated with 6-OHDA. Data are presented as mean ± SEM, n = 5–9, ^###<i>P</i><0.001 versus control; *<i>P</i><0.05, ***<i>P</i><0.001 versus 6-OHDA-treated cells; ^†<i>P</i><0.05, ^†††<i>P</i><0.001 versus ACS84-treated cells.</p

Effect of ACS84 on dopamine and its metabolites in 6-OHDA-lesioned striatum.

<p>The concentration of dopamine and its metabolites in 6-OHDA-lesioned striatum were measured using HPLC. Units for Dopamine, DOPAC and HVA concentrations were ng/g tissue. Data are presented as mean ± SEM, n = 6–8.</p>#<p>p<0.05 versus Sham group and *p<0.05 versus Vehicle group.</p