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Drug evaluation based on phosphomimetic PDHA1 reveals the complexity of
activity–related cell death in A549 non-small cell lung cancer cells
Ling Jin 1,2 , Minkyoung Cho 2 , Sung-Jin Bae 2 , Ki-Tae Ha 1,2,*
1 Department of Korean Medical Science, School of Korean Medicine, Pusan National University, Yangsan 50612, Korea;
2 Korean Medical Research Center for Healthy Aging, Pusan National University, Yangsan 50612, Korea;

ABSTRACT RESULTS

Cancer cells predominantly generate energy Figure 1. CRISPR/Cas9–mediated PDHA1
via glycolysis, even in the presence of knockout in A549 cells Figure 2. Construction and analyses of
oxygen, to support abnormal cell phosphomimetic PDHA1–expressing A549
proliferation. Suppression of PDHA1 by cells.
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
PDHA1 WT and PDHA1 3SD were transduced.
Since PDHA1 3SD cannot be phosphorylated
by PDKs, it was used to evaluate the
specific activity of PDK inhibitors. This study (A) Genomic DNA was extracted, and the target region of PDHA1
highlights that PDHA1 WT and PDHA1 3SD was amplified by polymerase chain reaction (PCR). PCR products
A549 cells can be used as a cell-based PDK were extracted and incubated with T7 Endonuclease Ⅰ. The
inhibitor–distinction system to examine the fragments are indicated by the red arrow. (B) Total PDHA1
expression was detected by western blot. HSP90 was used as an
relationship between PDH activity and cell internal control. (C) Sequencing analysis was performed with
death by established PDK inhibitors. genomic DNA derived from A549 PDHA1 KO (#1) cells. The sgRNA-
target sequence is marked with a red line; the PAM sequence is
Leelamine, huzhangoside A and marked with a blue line; the yellow and red arrows indicate the in/del
otobaphenol induced PDH activity- 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
dependent apoptosis, whereas AZD7545, with or without DCA (15 mM) for 24 h. PDHA1 activity was determined
VER-246608 and DCA effectively enhanced using a commercially available PDH activity kit. The relative activities
to DCA–free EV cells were calculated and are presented as means ±
PDHA1 activity but little toxic to cancer SEM. ***, p < 0.001.
cells. Furthermore, the activity of
phosphomimetic PDHA1 revealed the
complexity of its regulation, which requires Figure 3. Specificity assessments for targeting
PDH using PDHA1-manipulated A549 cells.
further in-depth investigation. (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
METHODS shown as means ± SEM. *, p < 0.05 compared with the KO control (1st
line), ###, p < 0.001 compared with the WT control (2nd line). (C, D) OCR
(C) and ECAR (D) in PDHA1 WT and PDHA1 3SD A549 cells were measured
CRISPR-mediated genome editing of PDHA1 with or without DCA (15 mM) as indicated. Values are presented as
means ± SD. .
Target sequences for PDHA1 were inspected by
CHOPCHOP provided by Zhang Lab (Cambridge,
MA). The sgRNA sequences for PDHA1 are 5’- Figure 4. Validations for PDH–dependent
GATGCAGACTGTACGCCGAA-3’ (exon3), 5’- apoptotic cell death using PDHA1-manipulated
GAAATGTGACCTTCACCGGC-3’ (exon4), 5’- A549 cells.
AGACAGCACGCGGGAGACGG-3’ (exon1), and
5’-TGTGCGTCCGAGAGGCAACA-3' (exon8).
Construction of phosphomimetic of PDHA1
PDHA1 3SD was carried out with a muta-direct site-
directed mutagenesis kit (iNtRON Biotechnology,
Seoul, Korea). Then, PDHA1 WT and PDHA1 3SD
were cloned into the pMX-IRES-HygromycinR
retroviral vector.
T7 Endonuclease Ⅰ assay
Genomic DNA was purified using a Genomic DNA
Prep Kit (SolGent, Daejeon, Korea). The PCR
amplifications were carried out, annealed and
incubated with 1 μL of T7 Endonuclease I (New
England Biolabs, Ipswich, MA) at 37°C for 15 min.
Nuclease-specific cleavage products were
determined by agarose gel electrophoresis. (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.
Cellular energy metabolism by Seahorse XFe96 (A) The levels of phosphorylated serines in PDHA1 (S232, S293 and
Analyzer S300) and total PDHA1 were examined by western blot analysis.
HSP90 was used as an internal control. (B–D) The densitometric
The OCR and ECAR were measured using an analyses (p-PDHA1/PDHA1) from three independent experiments were
XFe96 Extracellular Flux Analyzer (Seahorse performed and are presented by means ± SEM. *, p < 0.05, **, p < 0.01
Bioscience, Billerica, MA) according to the ***, p < 0.001. (E) PDHA1 WT and PDHA1 3SD A549 cells were treated PDHA1 WT (WT) and PDHA1 3SD (3SD) A549 cells were treated with DCA
with DCA (10 μM), JX06 (10 μM), AZD7545 (10 μM), VER-246608 (10
manufacturer’s protocol. μM), Leelamine (10 μM), huzhangoside A (1 μM) and otobaphenol (10 (50 mM; A), JX06 (50 μM; B), AZD7545 (20 μM; C), VER-246608 (50 μM;
μM) for 4 h as indicated. PDH activity was measured by a commercially 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
PDH activity assay available PDH activity assay kit. The relative activities to untreated iodide (PI) were analyzed by flow cytometry. (H) The percent frequency
PDHA1 WT cells were calculated and are presented as means ± SEM.
The PDH activity was measured using a ***, p < 0.001 compared with the WT control. of Annexin Ⅴ–positive apoptotic cells is shown as means ± SEM. **, p
commercial kit (MAK183; Sigma-Aldrich) according < 0.01; ***, p < 0.001.
to the manufacturer’s protocol.
CONCLUSION
Detection of apoptotic cells by flow cytometry
PDHA1 WT and PDHA1 3SD A549 cells were treated
with various concentrations of drugs for 24 h. PDHA1 WT and PDHA1 3SD A549 cells can be used as a cell-based PDK inhibitor–distinction
Apoptotic cells were examined using an apoptosis system to examine the relationship between PDH activity and cell death by established PDK
detection kit (BD Biosciences, San Jose, CA), inhibitors. Leelamine, huzhangoside A and otobaphenol induced PDH activity-dependent
detected by an Attune Acoustic Focusing apoptosis, whereas AZD7545, VER-246608 and DCA effectively enhanced PDHA1 activity but
Cytometer (Invitrogen), and analyzed using FlowJo little toxic to cancer cells. Furthermore, the activity of phosphomimetic PDHA1 revealed the
software.
complexity of its regulation, which requires further in-depth investigation.