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Andrographolide, a Novel Pyruvate Dehydrogenase Kinase Inhibitor,
Induces Apoptosis of Human Lung Cancer Cells
Yoonju Do 1,2 , EunSun Yang 2 , Jung-Suk Jin 2 , Kibong Kim 3 , 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;
3 Department of Pediatrics, Pusan National University Korean Hospital, Yangsan 50612, Korea;

ABSTRACT RESULTS
Most of cancer cells produce their energy Figure 2. Andrographolide reduced the lactate
through a high level of glycolysis followed by Figure 1. Cell viability assay in selected cell lines.
lactic acid fermentation even in the presence production but increased mitochondrial ROS and
mitochondrial damage.
of abundant oxygen. The pyruvate
dehydrogenase kinase (PDK) 1 is commonly
overexpressed in tumors and recognized as a
therapeutic target in cancer. Here, we report
Andrographolide (AG), a diterpenoid lactone
isolated from Andrographis paniculate, as a
novel PDK inhibitor through screening from
natural product library. AG has been reported
to anti-inflammatory, anti-viral, immune-
stimulant properties and induced apoptosis in
cancer cells. However, the precise
mechanism of AG induces apoptosis has not
yet been clearly elucidated. As a result of
checking the cell viability by MTT assay with
various cells with drugs, only AG showed
cytotoxicity correlated with PDK1 expression
among the 17 types of PDK1 inhibitors
searched. AG decreases the PDK1 activity in
both in vitro enzymatic kinase assay and
intracellular phosphorylation of PDHA1 in
H292 cells with high PDK1 expression than in
A549 cells. The production of lactic acid was
inhibited only in H292 cells. And,
mitochondrial damages and mitochondrial
ROS were increased. Apoptosis signals
increased. Collectively, AG suppressed the
viability of lung cancer cells through inhibiting (A) The H292 cells have high level of lactate production compared to A549
cells. The A549 cells with treated with AG have not decreased. However,
PDK1 activity and subsequently inducing the H292 cells with treated with AG have slightly decreased depending on
mitochondrial ROS-dependent apoptotic cell concentration. (B) The images show that level of Mitochondria ROS using
death. Mito-SOX. The intensity of H292 cells was increased depending on
concentration of AG for 5h. However, The A549 cells have state of intensity
compared to H292 cells. (C) The percentage of mitochondrial ROS (D)
METHODS (A) Difference of proteins (p-PDHA1, PDHA1, PDK1, LDHB, HSP90) A549 cells and H292 cells were treated with various concentration of AG for
expression of various cell lines for selected 5 cells through expression of 12 h. The images show that andrographolide induced damage of
Lactate production assay PDK1. The 4 of cells (H292, H522, HCT116, DLD-1) had high expression mitochondria in H292 cells than A549 cells were significantly increased
depend on concentrations. The intensity of H292 cells were decreased by
Lactate production in A549 cell and H292 cell were of PDK1 and the A549 cells had low expression of PDK1. (B-R) The AG compared to A549 cells. (E) The percentage of mitochondria membrane
measured with the lactate fluorometric assay kit viability of cells with various concentrations by 17 drugs for selecting drugs potential.
more inhibit cell growth. As a result, A549 cells have resistant than the
(Biovision, CA, USA). Pre-treatment with various other cells (H292, H522, HCT116, DLD-1) by AG.
concentrations of AG for 1h before media was Figure 4. Andrographolide induced apoptotic cell
replaced with phenol red- and serum-free RPMI
and incubated for an hour at 37°C. Next, 1μL of the Figure 3. Andrographolide reduced the death through mitochondrial pathway.
medium from each well was assessed by phosphorylation of PDHA1.
absorbance at 570 nm with the Spectramax M2
spectrofluoromeric (Molecular Devices).
Measurement of mitochondria ROS
The production of mitochondria ROS in cells was
determined using MitoSOX™ Red (Invitrogen). In
short, 1 μM MitoSOX™ Red was added to the cells
cultured in a conditioned medium and incubated at
37°C for 10 min. The fluorescence intensity was
analyzed using a fluorescence microscope (Zeiss
AX10 Imager M1; Carl Zeiss Microimaging,
Oberkochen, Germany). To quantify the
mitochondrial ROS, the fluorescence intensity of
each captured image was calculated using Image J
(NIH, MD, USA).
Mitochondrial depolarization assay
A549 cells and H292 cells were incubated in the
culture media containing various concentrations of
AND for 12 h. After pre-treatment, the cells were
incubated with 250 nM tetramethylrhodamine
methyl ester (TMRM; Thermo Fisher Scientific) for
30 min. The cells were washed 2 times with
phosphate-buffered saline (PBS). The images of
the samples were observed and captured using a
fluorescence microscope (Zeiss AX10 Imager M1; (A) The chemical structure of Andrographolide. (B-C) Treated with AG at (A-B) A549 cells and H292 cells were treated with the indicated
concentrations of AG for 24 h. Apoptosis significantly increased in A549 cells
indicated concentrations for 1 h and extracted p-PDHA1 (Ser293), PDHA1,
Carl Zeiss Microimaging, Oberkochen, Germany). HSP90. Expression of p-PDHA1 (Ser293) decreased depend on and H292 cells depending on concentration of AG. (C) The percentage of
To quantify the mitochondrial membrane potential, concentrations of AG in H292 cells and A549 cells. (D) scheme of invitro apoptotic cells. (D) The levels of proteins related with apoptosis, such as
the fluorescence intensity of each captured image PDK assay. (E) p-PDHA1 (Ser293 and Ser300) decreased depend on PARP, caspase-3 and -9, were examined and the GAPDH and HSP90 were
was calculated using Image J (NIH, MD, USA). concentration by inhibiting PDK1. used for internal controls.
Detection of apoptotic cells by flow cytometry CONCLUSION
A549 cells and H292 cells were treated with
various concentrations of AG for 24 h. Apoptosis
cells were examined using an annexin V-FITC The NCI-H292 cells are a continuous line with growth characteristics. Also, the genetic polyploidy of the cells
Apoptosis Detection Kit (Life Technologies, may limit the number of passages of usable cells. The H292 cells have high expression of PDK 1 and lactate
Carlsbad, CA, USA). The cells were seeded 2 X production compare with A549 cells. When Andrographolide was treated with human lung carcinoma cancer
10 5 / ml in 6 well plates and resuspended in 500 μL cells (NCI-H292), cell growth was inhibited. In addition, the phosphorylation of PDHA1 was decreased and
of binding buffer and incubated with 5 μL of lactate production were also decreased in H292 cells by Andrographolide. However, oxidative
annexin V-FITC and propidium iodine (PI) for 15 phosphorylation by reducing expression of PDK 1 induced the increment of mitochondrial ROS and
min at room temperature. The fluorescence mitochondrial membrane damage by Andrographolide was increased compared with A549 cells. These result
intensities of the samples were examined using a causes apoptosis of lung carcinoma cancer cells. In conclusion, I suggest that Andrographolide can be a
BD FACs Canto II flow cytometer by measuring the compound for development of novel drug treating lung cancer through inhibiting aerobic glycolysis and
annexin V-FITC excitation/emission at the inducing ROS-mitochondria mediated apoptotic cell death.
wavelengths of 280 nm.