30 research outputs found

    Challenges and Opportunities in the Development of Serine Synthetic Pathway Inhibitors for Cancer Therapy

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    Recent advances in the understanding of the relationship between cancer and metabolism have highlighted the relevance of the serine synthetic pathway (SSP), which consists of three successive enzymatic reactions. Enzymes of the SSP, such as phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase 1 (PSAT-1), were recently highlighted because they are amplified in a significant subset of human tumors, and their suppression by RNAi caused a decrease in cancer cell survival and growth. Currently, the discovery of drugs that inhibit these enzymes is still in its infancy, and the identification of suitable inhibitors could serve to understand the emerging biology of these metabolic enzymes. In this review, we present the SSP as a significant and novel emerging area for medicinal chemistry and we provide an overview of one of the key enzymes of the pathway, PHGDH

    MCT1 expression in tumor cells regulates lactate-induced angiogenesis and tumor growth.

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    <p>Two groups of BALB/c nude mice were injected s.c. with Matrigel plugs containing 30 mM lactate (right flank) or and equal volume of saline (left flank). The plugs also contained 10<sup>6</sup> SiHa TCs infected with a control shRNA (shCTR, Group 1) or 10<sup>6</sup> SiHa TCs infected with a specific shRNA against MCT1 (shMCT1-1, Group 2). (A) Tumor growth was tracked over time. The graph shows tumor growth curves and the pictures are representative of mice 21 days after tumor implantation. **<i>p</i><0.01; <i>ns</i>, <i>p</i>>0.05; <i>n</i> = 5 for Group 1; <i>n</i> = 4 for Group 2. (B) In a second set of experiments, plugs were microdissected 12 days after implantation. Pictures show CD31-positive endothelial cells (red) and α-smooth muscle actin-positive pericytes (green). Nuclei are stained in blue with DAPI. Bar = 50 µm. CD31 staining was quantified and is expressed as % of positive surface area in the graph. <i>n</i> = 3–4 for Group 1, <i>n</i> = 4–5 for Group 2. *<i>p</i><0.05.</p

    Lactate activates HIF-1 in normoxic oxidative tumor cells.

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    <p>(A) HIF-1 activity was quantified using a dual reporter luciferase assay in oxidative SiHa (left panel, <i>n</i> = 6–8), HeLa (middle panel, <i>n</i> = 3–4), and FaDu cancer cells (left panel, <i>n</i> = 3–4). All cells were cultured during 24-h in fresh medium containing 10 mM lactate or not (control). *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.005. (B) HIF-1α and β-actin protein expression was detected using Western blotting in the lysates of wild-type (WT, <i>n</i> = 3) or mitochondria-depleted (ρ0, <i>n</i> = 5–6) SiHa TCs cultured during 24-h in the presence of 10 mM lactate or not. The upper panels show representative experiments and the graphs HIF-1α protein expression normalized to β-actin levels. <i>ns</i>, <i>p</i>>0.05, **<i>p</i><0.01, ***p<0.005. (C–E) The level of <i>VEGF-A</i> transcript was determined using RT-qPCR in TC lysates. (C) SiHa TCs were exposed to 10 mM lactate or not during 24-h. **<i>p</i> = 0.009; <i>n</i> = 6. (D) As in (C) but with WiDr TCs. <i>ns</i>, <i>p</i>>0.05; <i>n</i> = 3. (E) SiHa TCs were cultured during 24-h in the presence of 10 mM lactate, 10 mM lactate+10 nM echinomycin (an inhibitor of the transcriptional activity of HIF-1), or none of these drugs (control). ***<i>p</i><0.005 <i>versus</i> control; <sup>###</sup><i>p</i><0.005 <i>versus</i> lactate condition; <i>n</i> = 3–4.</p

    Lactate inhibits PHD2 activity in oxidative tumor cells.

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    <p>(A) HIF-1α and β-actin protein expression was detected using Western blotting in the lysates of SiHa TCs incubated during 24-h with 10 mM lactate or not and increasing doses of 2-oxoglutarate. The upper panels show representative experiments and the graph HIF-1α protein expression normalized to β-actin levels. Data are expressed as % of lactate induction. **<i>p</i><0.01, ***<i>p</i><0.005 <i>versus</i> 10 mM lactate without 2-oxoglutarate; <i>n</i> = 8. (B) ODD-driven luciferase activity was measured in SiHa TCs treated during 24-h with 10 mM lactate or not. *<i>p</i> = 0.0206; <i>n</i> = 6. (C) HIF-1α and β-actin protein expression was detected using Western blotting in the lysates of SiHa TCs transfected with a specific siRNA against PHD2 (siPHD2) or with a control siRNA (siCTR) and incubated during 24-h with 10 mM lactate or not. The upper panels show representative experiments and the graph HIF-1α protein expression normalized to β-actin levels. <i>ns</i>, <i>p</i>>0.05, *<i>p</i><0.05; <i>n</i> = 3.</p

    Lactate induces normoxic HIF-1α protein stabilization in oxidative tumor cells, not in Warburg tumor cells.

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    <p>(A) Lactate release in the supernatant of SiHa and WiDr TCs was measured using a CMA600 enzymatic analyzer after 24-h of culture in fresh medium. ***<i>p</i> = 0.0002; <i>n</i> = 4. (B–G) HIF-1α and β-actin protein expression was detected using Western blotting in the lysates of oxidative SiHa or Warburg WiDr TCs. The upper panels show representative experiments and the graphs HIF-1α protein expression normalized to β-actin levels. (B) SiHa and WiDr cells were untreated to detect basal HIF-1α protein expression. *<i>p</i> = 0.0414; <i>n</i> = 3–4. (C) SiHa TCs were cultured during 24-h under hypoxia (1% O<sub>2</sub>) or not. *<i>p</i> = 0.011; <i>n</i> = 3. (D) As in (C) but with WiDr TCs. **<i>p</i> = 0.0057; <i>n</i> = 5. (E) SiHa TCs were cultured during 24-h in the presence of 10 mM lactate or not. **<i>p</i> = 0.0029; <i>n</i> = 4. (F) As in (E) but with WiDr TCs. <i>ns</i>, <i>p</i> = 0.1449; <i>n</i> = 8. (G) SiHa TCs were exposed to increasing doses of lactate during 24-h. *<i>p</i><0.05, **<i>p</i><0.01 <i>versus</i> 0 mM lactate condition; <i>n</i> = 9–11. (H) SiHa TCs were exposed to 10 mM lactate during increasing periods of time. *<i>p</i><0.05, **<i>p</i><0.01 <i>versus</i> time 0; <i>n</i> = 3.</p

    MCT1 and CD147 interact at the plasma membrane of oxidative tumor cells.

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    <p>(A) The left graph shows basal <i>SLC16A1</i>/MCT1 mRNA expression normalized to <i>RPL19</i> mRNA expression detected in SiHa and WiDr TCs using RT-qPCR. **<i>p</i> = 0.0062; <i>n</i> = 5–8. On the right, MCT1 and β-actin were detected using Western blotting (WB) in cell lysates. The upper panels show a representative experiment and the graph HIF-1α protein expression normalized to β-actin levels. ***<i>p</i> = 0.0004; <i>n</i> = 5. (B) as in (A) but detecting CD147 instead of MCT1. RT-qPCR: ***<i>p</i><0.0001; <i>n</i> = 7. WB: *<i>p</i> = 0.0237; <i>n</i> = 6. (C) as in (A) but detecting <i>LDH-B</i> and LDH-H instead of MCT1. RT-qPCR: **<i>p</i> = 0.008; <i>n</i> = 3. WB: ***<i>p</i><0.001; <i>n</i> = 6. (D) MCT1 (red, left upper panel) and CD147 (green, left medium panel) were detected using immunocytofluorimetry in SiHa TCs. The lower left panel is a merged picture in which cell nuclei have been stained in blue (Hoechst 33342). Right panels show control experiments in which primary antibodies were omitted. Pictures are representative of <i>n</i> = 4. Bar = 20 µm. (E) The <i>in situ</i> interaction between MCT1 and CD147 was verified in SiHa TCs using a proximity ligation assay. MCT1-CD147 interaction is identified with a red staining and cell nuclei are in blue (Hoechst 33342) in the left panel. The right panel shows control experiments in which the primary antibody against CD147 was omitted. Pictures are representative of <i>n</i> = 6. Bars = 10 µm.</p

    Characterization of endothelial dysfunction in ApoE-KO mice.

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    <p>(A) Acetylcholine (ACh) evoked relaxation in second branch of mesenteric arteries isolated from C57BL/6J and ApoE-KO mice after phenylephrine (Phe) contraction (10μM) in presence (filled) or absence (open) of indomethacin (10μM). Isolated relaxation pathways: (B) NO-mediated relaxation was evaluated in presence of indomethacin (10μM) after contraction with high-KCl solution (50mM). (C) EDH(F)-mediated relaxation was evaluated in presence of L-Ω-NoArg (100μM) and indomethacin (10μM) after contraction with phenylephrine (10μM). (D) PGI<sub>2</sub>-dependent relaxation was performed in presence of L-Ω-NoArg (100μM) after KCl (50mM) contraction. Results are expressed as mean ± SEM (N = 5–8 animals in each group). * P<0.05, ** P<0.01, *** P<0.001 ApoE-KO versus C57BL/6J mice, and # P<0.05 ApoE-KO (-Indo) versus ApoE-KO (+Indo)</p

    Effects of short <i>ex-vivo</i> and long <i>in-vivo</i> BM-573 treatment on the microvascular relaxation.

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    <p>(A, B and C) relaxation experiments were performed in microarteries isolated from 15-week-old mice preincubated in presence or absence of 3μM BM-573 during 1H (<i>ex-vivo</i>). (D, E and F) relaxation experiments were done in vessels isolated from 15-week-old mice that have been treated or not with BM-573 (10mg/L of drinking water) during 8 weeks. Acetylcholine-evoked relaxation was evaluated after phenylephrine or high-KCl solution (50mM) contraction to characterize: (A, D) Total-, (B, E) NO- or (C, F) EDH(F)-mediated relaxations in second branch of mesenteric arteries isolated from C57BL/6J and ApoE-KO mice treated or not with BM-573. Results are expressed as mean ± SEM (N = 5–8 animals in each group).* P<0.05, ** P<0.01 ApoE-KO versus C57BL/6J mice, and # P<0.05, ## P<0.01 ApoE-KO versus ApoE-KO+BM-573 treatment.</p

    Effects of BM-573 on NO availability and eNOS-derived production pathway.

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    <p>(A) Ionomycin (2μM) stimulated NO production was measured by EPR spin trapping in isolated aortic rings from ApoE-KO and C57BL/6J mice treated or not with BM-573. The EPR signals of accumulated [Fe(II)NO-(DETC)<sub>2</sub>] complex were normalized to dry tissue weight (N = 4–6 animals in each group). (B, C) NO bioavailability was measured as level of Hb-NO complex assayed by EPR spectroscopy in venous blood: (B) typical EPR spectra of Hb-NO, presented as high-field component of the signal (hfs) after subtraction of protein-centered free radical signal; (C) quantitation of Hb-NO in whole blood of ApoE-KO mice treated or not with BM573 (N = 6–12 animals in each group). (D, E) Phosphorylation of Akt (Ser 473) and eNOS (Ser1177) measured by western blot in aorta homogenates. Results are expressed as mean ± SEM (N = 8 animals in each group). * P<0.05 ApoE-KO versus C57BL/6J mice, δ P<0.05 C57BL/6J versus C57BL/6J+BM-573 treatment and # P<0.05 ApoE-KO versus ApoE-KO+BM-573 treatment.</p

    Effects of BM-573 on NOX-2, NOX-4 and COX-2 expression.

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    <p>Quantitative real-time PCR-based analysis of NOX-2 (A), NOX-4 (B) and COX-2 (C) expression in aortic homogenates from C57BL/6J and ApoE-KO mice treated or not with BM-573. (D) COX-2 protein expression assessed by western blotting. Results are expressed as mean ± SEM (N = 3–6 animals in each group). * P<0.05 ApoE-KO versus C57BL/6J mice and # P<0.05 ApoE-KO versus ApoE-KO+BM-573 treatment.</p
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