Mfrankfinal The Effects of Triptolide On Cellular Metabolism in Pancreatic Cancer and Fibroblast Cells

The Effects of Triptolide on Cellular Metabolism in Pancreatic
Cancer and Fibroblast Cells

Morgan
20
20
20
20
40
40
40
40
0.0
0
20
20
Time (min)
Time (min)
Time (min)
Time (min)
40
Time (min)
Time (min)
Time (min)
Time (min)
60
60
60
60
80
Glycolytic Function
of PS1
MIAPaca2
PS1
Untreated
6hrs (0.3M)
Untreated-MIAPaca2 20K
Glycolytic Function
Glycolytic Function
Triptolide 0.3uM 3hr-MIAPaca2 20K

Triptolide 0.3uM 4hr-MIAPaca2 20
GlycolyticTriptolide 0.3uM 2hr-MIAPaca2 20K

function inTriptolide 0.3uM 2hr-MIAPaca2 20K
MIAPaca-2
(A)Triptolide 0.3uM 4hr-MIAPaca2 20K
and
PS1 (B) after kinetic triptolide
150.0
150.0
150.0
treatment: significant response evident after 6-hours.
100.0
100.0
100.0 Triptolide 0.3uM 6hr-MIAPaca2 20K
50.0
Effect
of 0.3uM
Triptolide
on Cell Energy
50.0
150.0
150.0
50.0
150.0
Phenotypes
0.0
0.0
100.0
100.0
Glycolytic Function
50.0
MIAPaca2
PS1
0.0
6hrs (0.3M)
Untreated
Glycolytic Function
0
Time (min)
Cells left overnight to grow in a cell culture incubator
ECAR (mpH/min)
ECAR (mpH/min)
ECAR (mpH/min)

Significant
response is evident in mitochondrial function of MIAPaca-2
150.0 treatment.
150.0
150.0
(A) and PS1 (B) at 6Triptolide 0.3uM 2hr-MIAPaca2 20K
hours
of 0.3uMTriptolide 0.3uM 4hr-MIAPaca2 20K
triptolide
100.0
100.0
100.0
Kinetic Time Course

Experiment:
Cells pretreated with 0.3uM
concentration of triptolide for
varying times (0, 1, 2, 3, 4, 6 hrs.)
100.0 150.0
50.0 100.0
50.0
0.0
0.0
B)
ECAR (mpH/min)
Results
100.00.0
100.0
50.0
50.0
0
0.0
0.0
00
20
20
0.0
0.0
20
20
20
40 40
00
20
20
40
Time (min)
4040
00
Glycolytic
20 Capacity
20
4040
40
Time (min)
4040
60
Time (min)
Time (min)
6060
6060
6060
80
80
Time (min)
Time (min)
Time (min)
80
80
MIAPaca2
Untreated
PS1
Triptolide (3M)
Time (min)
Glycolytic Reserve
Glycolytic Function of
PS1
200.0
150.0
100.0
50.0
0.0
100.00.0
Untreated:
Baseline
50.0
50.0
0.0
0.0
150.0
MIAPaca-2
150.0
50.0
100.00.0
100.0
50.0
50.0
0.0
0.0
20
0
00
20
20
20
40
Time (min)
Stressed
0.3uM Triptolide:
Stressed
Baseline
0 B)
00
20
20
20
4040
PS1
40
Time (min)
20
20
4040
40
Time (min)
4040
00
60
80
Time (min)
Time (min)
6060
60
80
Time (min)
Time (min)
6060
606
8080
8080
60
80
Time (min)
Time (min)
Metabolic Potential
Untreated:
Baseline
Stressed
Metabolic Potential
Metabolic Potential
0.3uM Triptolide:
Stressed
Baseline
Metabolic Potential
The Phenotype Stress Test (PST) shows that triptolide (0.3uM)

treatment reduces the overall metabolic potential of PS1 cells (B) but
6060not MIAPaca-2
8080
(A). MIAPaca-2 and PS1 both become more glycolytic
80
after triptolide (0.3uM) treatment.
8080
60
80
Time (min)
Time (min)
ECAR (mpH/min)
200.0
20
ECAR (mpH/min)
ECAR (mpH/min)
ECAR (mpH/min)
50.0
0
50.0
150.0
150.050.0MIAPaca-2
A)
150.0
ECAR (mpH/min)
ECAR (mpH/min)
GST:
MST:
to determine
to determine
when 0.3uM
when 0.3uM
triptolide starts
triptolide starts
to have a
to have a
significant effect significant effect
on glycolytic
on mitochondrial
function of
function of
MIAPaca-2 and MIAPaca-2 and
PS1 cells
PS1 cells
ECAR (mpH/min)
Dose Response
of
Triptolide
Effect
on
50.0
50.0
150.0
150.0
50.0
Glycolysis0.0
0.0
100.0
100.0
0.0
100.0
6hrs (0.3M)
Untreated
Glycolytic Function
Time (min)

ECAR (mpH/min)
20,000 cells seeded per well on a 96-well cell culture plate
MIAPaca2
PS1
Glycolytic Function
20
40
60
80
Glycolytic Function
A)
Glycolytic Function 150.0
Glycolytic Function
0
20
40
60
80
Glycolytic Function
Glycolytic Function
Spare Respiratory
Capacity
OCR (pmol/min)
OCR (pmol/min)
Cells harvested, re-suspended, and counted
80.0
60.0
40.0
20.0
0.0
Glycolytic Function
Glycolytic Function
50.0
Glycolytic Function
Glycolytic Function
PS1
Glycolytic Function
100.0
Time (min)
B) Mitochondrial Respiration of
Glycolytic Function
Glycolytic Function
150.0
80
8080
80
80
8080
80
60
6hrs (0.3M)
Untreated
Glycolytic Reserve
Reserve
Glycolytic
ECAR (mpH/min)
ECAR (mpH/min)
0.0100.0
0.0
0.0
0.0
0
0
0
50.0
60
60
60
60
80
PS1
Time (min)
ECAR (mpH/min)
150.0
40
40
40
40
60
MIAPaca2
ECAR (mpH/min)
ECAR (mpH/min)
ECAR (mpH/min)
ECAR (mpH/min)
ECAR (mpH/min)
ECAR (mpH/min)
Mitochondrial0.0
Respiration of
0.0
0.0
0.0
MIAPaca-2
20
00
20
20
40
60
80
60
60
60 0.0 8080
80
Time (min)
Time (min)
Time (min)
ATP Production
Time (min)
OCR (pmol/min)
100.0
100.0
100.0
100.0
ECAR (mpH/min)
ECAR (mpH/min)
ECAR (mpH/min)
ECAR (mpH/min)
A)
OCR (pmol/min)
ECAR (mpH/min)
ECAR (mpH/min)
ECAR (mpH/min)
ECAR (mpH/min)
Kinetic Dosing of Triptolide0.0

Effect on Oxidative
0.0
0.0
Triptolide 3uM 6hr-PS1 20K
0.0
0
20
40
100.0Phosphorylation
100.0
100.0
0
20
40
20
40
100.0
0
20
40
200.0
200.0
200.0
20
B)
100.0
ECAR (mpH/min)
Triptolide 0.1uM 6hr*-PS1 20K

Untreated-PS1 20K
100.0
MIAPaca2
PS1
Untreated-PS1 20K
0
20
40
60
80
Triptolide (3M)
Untreated
Glycolytic Function
50.0
Triptolide 0.3uM 6hr-PS1 20K
Time (min)
0.0
Triptolide 1uM 6hr*-PS1 20K
Untreated-PS1 20K
Untreated-PS1 20K
0
Glycolytic Function
Glycolytic
Glycolytic Capacity
Capacity
150.0
ECAR (mpH/min)
ECAR (mpH/min)
0.0
ECAR (mpH/min)
Glycolytic Function
Glycolytic Function
100.0
MIAPaca-2
A)
Untreated-PS1 20K
Untreated-PS1 20K
ECAR (mpH/min)
ECAR (mpH/min)
ECAR (mpH/min)
200.0

200.0
200.0
200.0
Cells grown in a T75 flask to 70-80% confluency
GST:
to determine
the minimum
dose that elicits
a significant
response in
glycolysis of
MIAPaca-2 and
PS1 cells
OCR (pmol/min)
OCR (pmol/min)
300.0
Kinetic Dosing of Triptolide Effect on

Glycolysis
Glycolytic Function
Glycolytic Function
200.0
Dose dependent
response is evident
in
mitochondrial function of
200.0
200.0
MIAPaca-2 (A) and PS1 (B). The minimal dose that elicits significant
response compared to control isTriptolide 3uM 6hr-PS1 20K
0.3uM.
100.0
100.0
100.0
Experimental design:
Dose Response Experiment:

Cells pretreated with increasing
concentrations of triptolide (0uM,
0.03uM, 0.1uM, 0.3uM, 1uM, 3uM)
for total of 6 hours
Spare Respiratory
Capacity
PS1
Glycolytic Function
Glycolytic Function
Conclusion
Triptolide adversely affects glycolytic and mitochondrial
function in both pancreatic cancer cells and fibroblasts.
Acknowledgements
This work was funded by Helios Education Foundation.

Dose Response of Triptolide Effect on
We wish to thank the members of the Pancreatic Cancer
Glycolytic Function
Glycolytic Function
Triptolide 0.03uM 6hr*-PS1 20KResearch Lab for their technical support: Ruben Munoz,
Oxidative Phosphorylation
Serina Ng, Maria Nicolas-Perez, Jennifer Harper, and
Untreated-PS1 20K
A) Mitochondrial Respiration of
Untreated-PS1 20K
ATP Production
MIAPaca2
PS1
0Glycolytic Function
20
40
60
80
Tiffanie Capello Lee.
Triptolide (3M)
MIAPaca-2
Glycolytic Function Triptolide 0.03uM 6hr*-PS1 20K
Untreated
Time (min)Triptolide 0.03uM 6hr*-PS1 20K
We would also like to thank George Rogers and Andy
200.0
Untreated-PS1 20K
Triptolide 1uM 6hr*-PS1 20K Nelson with Agilent Technologies for their help with data
Untreated-PS1 20K
150.0
100.0
interpretation.
50.0
Finally,
we
would
like
to
thank
Julie
Euber,
Brandy
Wells,
Dose
dependent
response
is
evident
in
glycolytic
function
of
200.0
200.0
0.0
200.0
MIAPaca2
PS1
and Stephanie Gomez from TGen Education and
MIAPaca-2
(A)
and
PS1
(B).
The
minimal
dose
that
elicits
significant
0
20
40
60
80
Triptolide (3M)
Untreated
response
compared
to
control
is
0.3uM.
Outreach for their endless support and encouragement.
100.0
Time (min)
100.0
100.0
100.0
200.0
200.0
200.0
0.0
0.0
0.0
0.0
0
20
40
60
80
100.0
100.0
100.0
0
20
40
60
20
40
60
8080
100.0
0
20
40
60
80
200.0
200.0
1. Ansari, D., Tingstedt, B., Andersson, et al. (2016). Pancreatic cancer: Yesterday,
today and tomorrow. Future Oncology (London, England).
200.0
Time (min)
Time (min)
Time (min)
0.0
Time (min)
0.0
2. Chugh, R., Sangwan, V., Patil, S. P et al. (2012). A preclinical evaluation of minnelide as a therapeutic
agent
against
pancreatic
cancer.
Science Translational Medicine, 4(156), 156ra139.
0.0
0.0
100.0
20
40
60
80
100.0
3. Agilent Seahorse XF Technologies. (2016). Seahorse overview. Retrieved
from http://www.agilent.com/en-us/promotions/xftechnologyoverview.
100.0
000
20
40
60
20
40
60
8080
100.0
20
40
60
80
Time (min)
Time (min)
Time (min)
0.0
Time (min)
0.0
0.0
0.0
20
40
60
80
000
20
40
60
20
40
60
8080
20
40
60
80
Translational Genomics Research Institute | 445 N. 5th Street Phoenix,
AZ 85004 www.tgen.org
Time (min)
Time (min)
Time (min)
Time (min)
ECAR (mpH/min)
ECAR (mpH/min)
ECAR (mpH/min)
ECAR (mpH/min)
ECAR (mpH/min)
ECAR (mpH/min)
ECAR (mpH/min)
ECAR (mpH/min)
Conclusions
and
Future
Directions:
Triptolide adversely affects glycolytic and
mitochondrial function in pancreatic cancer
cells and fibroblasts. Future research should
include similar experiments using other PC cell
lines and correlate the effects on mitochondrial
and glycolytic function to anti-tumor activities to
provide a better understanding of the drugs
mechanism of action. Probing cellular energy
pathways in cancer cells treated with triptolide
in combination with current treatment options
and/or other new agents may lead to a better
alternative to treat PC.
Haiyong
Results
B) Mitochondrial Respiration of
ECAR (mpH/min)
ECAR (mpH/min)
ECAR (mpH/min)
ECAR (mpH/min)
Results: Our data suggests that triptolide

reduces
mitochondrial
respiration
and
glycolysis in a dose dependent manner in
MIAPaCa-2 and PS1 cells within six hours of
treatment. Under the tested conditions, 0.3uM
triptolide significantly reduced OCR and ECAR
in both cell lines. This response is evident in
key parameters of mitochondrial function (ATP
production, maximal respiration, and spare
respiratory capacity) and glycolytic function
(glycolytic capacity and glycolytic reserve).
2
Han
Results
Methods
MST:
to determine
the minimum
dose that elicits
a significant
response in
mitochondrial
respiration of
MIAPaca-2 and
PS1 cells
Daniel D. Von
OCR (pmol/min)
Methods: The Seahorse XFe96 Analyzer was

employed to probe cellular energy pathways
including
glycolysis
and
oxidative
phosphorylation in MIAPaca-2 and PS1. The
XF Cell Mito Stress Test (MST) measures
oxygen consumption rate (OCR) to evaluate
mitochondrial function and the XF Cell
Glycolysis Stress Test (GST) detects changes
in extracellular acidification rate (ECAR) to
evaluate glycolytic function. Dose response
and kinetic time course experiments were
performed on triptolide treated MIAPaca-2 and
PS1 cells. This was done to determine the
minimal dose that elicits a significantly different
response in oxidative phosphorylation and
glycolysis as well as its time course of action
when compared to control. Cells pretreated
with different concentrations of triptolide
(0.03uM to 3uM) for varying times up to six
hours were subjected to the GST or MST.
2
Hoff ,
Scholar Program at TGen, 2Clinical Translational Research Division, Translational Genomics Research Institute
Most cancer cells predominantly produce energy via

aerobic glycolysis. This is known as the Warburg
Effect.
The Seahorse Extracellular Flux Analyzer measures
mitochondrial respiration and glycolysis in live cells.
Mitochondrial respiration: cells consume oxygen to
produce ATP
Mito Stress Test (MST) measures mitochondrial
function via oxygen consumption rate (OCR)3
Key parameters: ATP production (energy production)
and spare respiratory capacity (cells ability to
respond to energy demand)
Glycolysis: cells convert glucose to pyruvate with the
concurrent production of ATP
Glycolysis Stress Test (GST) measures glycolytic
function via extracellular acidification rate (ECAR)3
Key parameters: glycolytic capacity (maximum
ECAR reached by a cell) and glycolytic reserve
(cells ability to respond to energy demand)
Energy phenotype: measures cells use of major
energy pathways as well as their metabolic potential
Metabolic potential shows the cells ability and
preferred pathway to respond to energy demand
OCR (pmol/min)
Objective: Targeting the mitochondrial and

glycolytic cellular energy pathways is an
effective way to inhibit tumor cell growth.
Therefore, this project aimed at elucidating the
effect of triptolide on the cellular energetics of
PC (MIAPaca-2) and fibroblast (PS1) cells,
which predominate the TME.
Pawan
Introduction
Abstract
Background: Pancreatic cancer (PC) is the
third leading cause of adult cancer death in the
United States and each year approximately
41,800 patients die from the disease. Late
diagnosis, limited treatment options, and low
response rates to current standard of care
make PC extremely lethal with devastatingly
low survival rates (>94% of patients die within
5 years of diagnosis)1. One of the primary
reasons for the dismal prognosis is that the
tumor microenvironment (TME) in PC is
incredibly dense, fibrotic, and hypo-vascular
making it impenetrable to chemotherapeutics1.
Triptolide (Minnelide), a compound with antitumor activity, is in Phase I clinical trials to treat
various solid tumors and is showing promising
activity in several cancer types including PC2.
2
Noel ,
ECAR (mpH/min)
ECAR (mpH/min)
1Helios
1,2
Frank ,
References
Untreated-PS1 20K
Untreated-PS1 20K

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The Effects of Triptolide on Cellular Metabolism in Pancreatic

Cancer and Fibroblast Cells

Triptolide 0.3uM 3hr-MIAPaca2 20K

GlycolyticTriptolide 0.3uM 2hr-MIAPaca2 20K

Cells left overnight to grow in a cell culture incubator

Triptolide 0.3uM 1hr-MIAPaca2 20K

Kinetic Time Course

The Phenotype Stress Test (PST) shows that triptolide (0.3uM)

Triptolide 0.3uM 1hr-MIAPaca2 20K

20,000 cells seeded per well on a 96-well cell culture plate

Cells harvested, re-suspended, and counted

Kinetic Dosing of Triptolide0.0

Triptolide 0.1uM 6hr*-PS1 20K

Triptolide 1uM 6hr*-PS1 20K

Cells grown in a T75 flask to 70-80% confluency

Kinetic Dosing of Triptolide Effect on

Dose Response Experiment:

This work was funded by Helios Education Foundation.

Results: Our data suggests that triptolide

Methods: The Seahorse XFe96 Analyzer was

Most cancer cells predominantly produce energy via

Objective: Targeting the mitochondrial and

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