[논문]Drug evaluation based on phosphomimetic PDHA1 reveals the complexity of activity-related cell death in A549 non-small cell lung cancer cells

Jin, Ling; Cho, Minkyoung; Kim, Bo-Sung; Han, Jung Ho; Park, Sungmi; Lee, In-Kyu; Ryu, Dongryeol; Kim, Jae Ho; Bae, Sung-Jin; Ha, Ki-Tae

doi:10.5483/bmbrep.2021.54.11.101

[국내논문] Drug evaluation based on phosphomimetic PDHA1 reveals the complexity of activity-related cell death in A549 non-small cell lung cancer cells 원문보기

BMB reports, v.54 no.11, 2021년, pp.563 - 568

Jin, Ling (Department of Korean Medical Science, School of Korean Medicine, Pusan National University) , Cho, Minkyoung (Korean Medical Research Center for Healthy Aging, Pusan National University) , Kim, Bo-Sung (Department of Korean Medical Science, School of Korean Medicine, Pusan National University) , Han, Jung Ho (Department of Korean Medical Science, School of Korean Medicine, Pusan National University) , Park, Sungmi (Department of Internal Medicine, School of Medicine, Kyungpook National University) , Lee, In-Kyu (Department of Internal Medicine, School of Medicine, Kyungpook National University) , Ryu, Dongryeol (Department of Molecular Cell Biology, School of Medicine, Sungkyunkwan University) , Kim, Jae Ho (Department of Physiology, College of Medicine, Pusan National University) , Bae, Sung-Jin (Korean Medical Research Center for Healthy Aging, Pusan National University) , Ha, Ki-Tae (Department of Korean Medical Science, School of Korean Medicine, Pusan National University)

Abstract ▼ AI-Helper

Cancer cells predominantly generate energy via glycolysis, even in the presence of oxygen, to support abnormal cell proliferation. Suppression of PDHA1 by PDK1 prevents the conversion of cytoplasmic pyruvate into Acetyl-CoA. Several PDK inhibitors have been identified, but their clinical applications have not been successful for unclear reasons. In this study, endogenous PDHA1 in A549 cells was silenced by the CRISPR/Cas9 system, and PDHA1WT and PDHA13SD were transduced. Since PDHA13SD cannot be phosphorylated by PDKs, it was used to evaluate the specific activity of PDK inhibitors. This study highlights that PDHA1WT and PDHA13SD A549 cells can be used as a cell-based PDK inhibitor-distinction system to examine the relationship between PDH activity and cell death by established PDK inhibitors. Leelamine, huzhangoside A and otobaphenol induced PDH activity-dependent apoptosis, whereas AZD7545, VER-246608 and DCA effectively enhanced PDHA1 activity but little toxic to cancer cells. Furthermore, the activity of phosphomimetic PDHA1 revealed the complexity of its regulation, which requires further in-depth investigation.

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그림 Fig. 1. CRISPR/Cas9-mediated PDHA1 knockout in A549 cells. PDHA1- targeting lentiCRISPR vectors were transiently transfected into A549 cells as indicated. Untransfected and empty vector-transfected A549 cells (CON and EV, respectively) were used as a negative control. (A) Genomic DNA was extracted, and the target region of PDHA1 was amplified by polymerase chain reaction (PCR). PCR products were extracted and incubated with T7 Endonuclease I. The fragments are indicated by the red arrow. (B) Total PDHA1 expression was detected by western blot. HSP90 was used as an internal control. (C) Sequencing analysis was performed with genomic DNA derived from A549 PDHA1^KO (#1) cells. The sgRNA-target sequence is marked with a red line; the PAM sequence is marked with a blue line; the yellow and red arrows indicate the in/del mutation and cleavage site in A549 PDHA1^KO (#1) cells, respectively. (D) EV and PDHA1^KO (#1, #2, #3 and #4) A549 cells were treated with or without DCA (15 mM) for 24 h. PDHA1 activity was determined using a commercially available PDH activity kit. The relative activities to DCA-free EV cells were calculated and are presented as means ± SEM. ***P < 0.001.
그림 Fig. 2. Construction and analyses of phosphomimetic PDHA1-expressing A549 cells. PDHA1^WT or phosphomimetic PDHA1^3SD were reintroduced into PDHA1^KO A549 cells. (A, B) PDHA1^KO, PDHA1^WT and PDHA1^3SD A549 cells were treated with DCA (15 mM) for 24 h as indicated. (A) The levels of phosphorylated serines in PDHA1 (S232, S293 and S300), and total PDHA1 were examined by western blot analysis. β-ACTIN was used as an internal loading control. (B) PDH activity was measured by a commercially available PDH activity assay kit. The relative activities on DCA-free PDHA1^KO A549 cells were calculated and are shown as means ± SEM. *P < 0.05 compared with the KO control (1^st line), ^###P < 0.001 compared with the WT control (2^nd line). (C, D) OCR (C) and ECAR (D) in PDHA1^WT and PDHA1^3SD A549 cells were measured with or without DCA (15 mM) as indicated. Values are presented as means ± SD.
그림 Fig. 3. Specificity assessments for targeting PDH using PDHA1-manipulated A549 cells. (A-D) PDHA1^WT A549 cells were treated with DCA (10 μM), JX06 (10 μM), AZD7545 (10 μM), VER-246608 (10 μM), Leelamine (10 μM), huzhangoside A (1 μM) and otobaphenol (10 μM) for 4 h as indicated. (A) The levels of phosphorylated serines in PDHA1 (S232, S293 and S300) and total PDHA1 were examined by western blot analysis. HSP90 was used as an internal control. (B-D) The densitometric analyses (p-PDHA1/PDHA1) from three independent experiments were performed and are presented by means ± SEM. *P ＜ 0.05, **P ＜ 0.01 ***P ＜ 0.001. (E) PDHA1^WT and PDHA1^3SD A549 cells were treated with DCA (10 μM), JX06 (10 μM), AZD7545 (10 μM), VER-246608 (10 μM), Leelamine (10 μM), huzhangoside A (1 μM) and otobaphenol (10 μM) for 4 h as indicated. PDH activity was measured by a commercially available PDH activity assay kit. The relative activities to untreated PDHA1^WT cells were calculated and are presented as means ± SEM. ***P < 0.001 compared with the WT control.
그림 Fig. 4. Validations for PDH-dependent apoptotic cell death using PDHA1-manipulated A549 cells. PDHA1^WT (WT) and PDHA1^3SD (3SD) A549 cells were treated with DCA (50 mM; A), JX06 (50 μM; B), AZD7545 (20 μM; C), VER-246608 (50 μM; D), Leelamine (10 μM; E), huzhangoside A (0.1 μM; F) and otobaphenol (10 μM; G) for 24 h. The cells stained with Annexin Ⅴ and propidium iodide (PI) were analyzed by flow cytometry. (H) The percent frequency of Annexin Ⅴ-positive apoptotic cells is shown as means ± SEM. **P < 0.01; ***P < 0.001.

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