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Potential mechanisms of
ascorbate-induced cytotoxicity in
pancreatic cancer cells
Juan Du, Garry R. Buettner, Larry W. Oberley,
Joseph J. Cullen
From the Departments of Surgery, Radiation
Oncology and Free Radical & Radiation Biology
Program, University of Iowa College of
Medicine and VAMC, Iowa City, IA.
Abstract: Pharmacological concentrations of ascorbate easily achieved in humans may be effective in
cancer therapeutics (PNAS, 104:8749, 2007). We hypothesized that ascorbate concentrations achievable
with intravenous dosing may be cytotoxic in pancreatic cancer where the five year survival is < 3%.
Pancreatic cancer cell lines were treated with ascorbate (0, 5, and 10 mM) for one hour and viability and
clonogenic survival were determined. In addition, the immortal H6c7 cell line (pancreatic ductal
epithelial cell) and its derivatives, H6c7eR-pBp (retroviral vector control), H6c7er-Kras (H6c7 cells
expressing K-ras oncogene), and H6c7eR-KrasT (tumorigenic H6c7 cells expressing K-ras oncogene)
(Cancer Res., 65:5045, 2005) were treated with ascorbate (0, 5, and 10 mM) and viability were
determined. MIA PaCa-2 pancreatic cancer cell lines with functional mitochondria (rho +) and the same
lines without functional mitochondria (rhoo) (J. Biol. Chem. 281:37416, 2006), were treated with
ascorbate and clonogenic survival determined. The oxygen electrode method was used to determine
H2O2 production. There was a time and dose-dependent increase in measured H2O2 production with
increased concentrations of ascorbate. Ascorbate decreased clonogenic survival and viability in
pancreatic cancer cell lines in a dose-dependent manner. Ascorbate had no effect on the H6c7 cell line,
but decreased viability in the H6c7 cell lines that express K-ras oncogene. Ascorbate (5 and 10 mM)
decreased viability in all human pancreatic cancer cell lines tested. In rho + cells, ascorbate resulted in a
dose-dependent decrease in clonogenic survival, but no cytotoxicity in the rho o cells. We conclude that
pharmacological doses of ascorbate, achievable in humans when given intravenously, may have potential
for therapy in pancreatic cancer. The ascorbate-induced cytotoxicity in pancreatic cancer cells may be
mediated by a mitochondrial mechanism. Support: NIH grants CA115785, CA66081, the Medical
Research Service, Department of Veterans Affairs, and the Susan L. Bader Foundation of Hope.
Introduction
• Pharmacological concentrations of
ascorbate easily achieved in humans may be
effective in cancer therapeutics (PNAS,
104:8749, 2007).
Predicted plasma Vitamin C concentrations in healthy persons after oral (top) or intravenous
(bottom) administration of Vitamin C.
Annals of Internal Medicine 140:533, 2004
•
•
•
•
Ascorbate as an anti-tumor agent. Ascorbate (vitamin C, ascorbate) is one of the early unorthodox
therapies for cancer without supporting data. Initial published case reports demonstrated potential
benefit from high dose ascorbate treatment. Subsequent reports documented the results of 100 patients
with terminal cancer, in whom conventional therapy was no longer considered useful and were given
intravenous ascorbate. Patients who received ascorbate survived 300 days longer than controls. A
prospective study was then conducted randomizing patients to ascorbate treatment or palliative therapy.
Treated patients had a median survival of 343 days vs. 180 days for controls. Smaller studies have also
reported benefits of ascorbate.
To test whether ascorbate was effective, Moertel conducted two randomized placebo controlled studies
randomized to oral ascorbate and neither study showed any benefit. Because Moertel’s studies were
taken as definitive, ascorbate treatment was considered useless. However Moertel’s results were not
comparable to those previous studies because ascorbate was given orally and not intravenously.
Emerging knowledge suggests that the role of ascorbate in cancer treatment should be reexamined. The
evidence falls into two categories: clinical data on dose concentration relationships and laboratory data
describing potential cell toxicity with high concentrations of ascorbate in vitro. Clinical data show that
when ascorbate is given orally, fasting plasma concentrations are tightly controlled at < 100 M. As
doses exceed 200 mg, absorption decreases, urine excretion increases and ascorbate bioavailability is
reduced. In contrast, when 1.25 grams of ascorbate are administered intravenously, concentrations as
high as 1 mM are achieved. Some clinicians have infused more than 10 grams of ascorbate in cancer
patients and achieved plasma concentration of 1 to 5 mM. Thus, it is clear that intravenous
administration of ascorbate can yield very high plasma levels, while oral treatment does not.
Chen et al. measured cell death in 10 cancer and 4 normal cell types using 1 hour exposures to
ascorbate. Normal cells were unaffected by 20 mM ascorbate whereas 5 cancer cell lines had EC50
values of < 4 mM, a concentration achievable by intravenous administration. In addition, cell death was
independent of metal chelators and dependent on H2O2 formation. H2O2 generation was dependent on
ascorbate concentration, incubation time, and displayed a linear relationship with ascorbate radical
formation. In vivo, Chen and colleagues demonstrated that intravenous injection of ascorbate (0.25-0.5
mg/g body weight) increased baseline concentrations of ascorbate in blood and extracellular fluid to >
8 mM and increased formation H2O2. These studies provides a foundation for pursuing pharmacologic
ascorbate as a prooxidant agent in cancer therapy.
J. Am. Coll. Nutrition 19:423, 2000
• Pancreatic cancer therapy. Pancreatic cancer is the 4th most common
cause of cancer death in the United States with over 33,000 fatal cases
annually in the United States alone. Surgical resection of the primary tumor
remains the only potentially curative treatment for pancreatic cancer.
However, in population-based studies the number of patients undergoing
resection with curative intent can be less than 3%. Even after resection,
median survival is only 12-18 months and less than 20% of resected patients
survive 5 years. The majority of patients die of metastatic cancer recurrence.
• Other adjuvant treatments such as radiation therapy and chemotherapy, have
not improved long-term survival after resection. The rate of
chemotherapeutic response is less than 20%, while less than 10% of patients
benefit from radiation therapy.
• Because of the lack of poor therapeutic responsiveness of pancreatic cancer
to surgery, chemotherapy, and radiation therapy, survival beyond five years
is rare with median survival less than six months.
• Thus, novel and effective therapies directed against pancreatic cancer are
needed to control progression and metastatic disease.
CA Cancer J Clin 57:43-66, 2007.
Cur Probl. Surg 36:59-152, 1999.
Hypothesis
• Ascorbate concentrations achievable with
intravenous dosing may be cytotoxic in
pancreatic cancer where the five year
survival is < 3%.
Methods
• Pancreatic cancer cell lines were treated with ascorbate (0, 5,
and 10 mM) for one hour and viability and clonogenic survival
were determined.
• The immortal H6c7 cell line (pancreatic ductal epithelial cell)
and its derivative H6c7er-Kras (H6c7 cells expressing K-ras
oncogene) (Cancer Res., 65:5045, 2005) were treated with
ascorbate (0, 5, and 10 mM) and viability were determined.
• MIA PaCa-2 pancreatic cancer cell lines with functional
mitochondria (rho+) and the same lines without functional
mitochondria (rhoo) (J. Biol. Chem. 281:37416, 2006), were
treated with ascorbate and clonogenic survival determined.
• The oxygen electrode method was used to determine H2O2
production.
(Clonogenic survival relative to 0 mM Ascorbate)
Clonogenic Survival
Mia PaCa-2
*P<0.05 vs 0 mM Ascorbate
Arrow indicates that no colonies
were formed when 20 mM ascorbate was
given for one hour
1
0.1
*
*
5 mM
10 mM
0.01
0.001
0 mM
Ascorbate
20 mM
(Clonogenic survival relative to 0 mM Ascorbate)
Clonogenic Survival
AsPC-1
*P<0.05 vs 0 mM Ascorbate
1
*
0.75
*
0.5
*
0.25
0
0 mM
5 mM
Ascorbate
10 mM
20 mM
Figure 1. MIA PaCa-2 and AsPC-1 pancreatic
cancer cells were treated with ascorbate (020 mM) for one hour and clonogenic
survival determined. Ascorbate caused a
dose-dependent decrease in clonogenic
survival in pancreatic cancer cell lines.
)
)
Baseline
Actin
Actin
Actin
MnSOD
GPx1
PhGPx
CuZnSOD
Trx
D.
1.8
rho (o)
rho (+)
1.2
0.6
0
AntA
+
Rh
o (o
)
Rh
o
Rh
o+
Rh
o (o
)
Rh
o+
Rh
o (o
)
GAPDH
relative to rho (o)
+
Rh
o (o
Rh
o
Rh
o (o
+
mtDNA
Fold increase in mean fluorescence intensity
B.
Rh
o
M
A.
C.
Figure 2. MIA PaCa-2 rho (o) cells depleted of mitochondrial DNA (mtDNA) were
generated by incubating wild type cells (rho +) for 6-8 weeks with 100 ng/ml ethidium
bromide. The medium was supplemented with 50 g/ml uridine and 100 g/ml
pyruvate to compensate for the respiratory metabolism deficit. After selection, the
MIA PaCa-2 rho (o) cells were cultured in the same medium without ethidium
bromide.
A. To verify mtDNA depletion, total cellular DNA was extracted and subjected to
PCR using two pairs of human mtDNA specific primers: 1) Mts1 (forward) (5cctagggataacagcgcaat-3) and Mtas 1 (reverse) (5 -tagaagagcgatggtgagag-3), which
gave a 630-bp product, and 2) Mts2 (forward) (5-aacatacccatggccaacct-3) and Mtas2
(reverse) (5-ggcaggagtaatcagaggtg-3), which gave a 532-bp product. For control, we
measured the expression of GAPDH, which is coded by nuclear DNA.
B. Immunoblot of cytochrome c demonstrating that this protein which is coded by
mtDNA is present in rho (+) but not in rho (o) cells.
C. Immunoblots for manganese superoxide dismutase (MnSOD), copper/zinc SOD
(CuZnSOD), thioredoxin (Trx), glutathione peroxidase (GPx1), and phospholipid
glutathione peroxidase (PhGPx).
D. Cells were incubated with DMSO and DMSO containing Antimycin A (AntA) 10
M for 15 minutes. Cells were stained for hydroethidine (DHE) and fluorescence
measured by flow cytometry. Mean fluorescence intensity (MFI) calculated relative to
rho (o) cells. Means  SEM, N = 3.
(Clonogenic survival relative to 0 mM Ascorbate)
Clonogenic Survival
Mia PaCa-2 rho+ cells
*P<0.05 vs 0 mM Ascorbate
1
*
0.75
*
0.5
0.25
*
0
0 mM
0.1 mM
Ascorbate
1 mM
5 mM
(Clonogenic survival relative to 0 mM Ascorbate)
Clonogenic Survival
Mia PaCa-2 rhoo cells
*P<0.05 vs 0 mM Ascorbate
1
0.75
0.5
0.25
0
0 mM
0.1 mM
Ascorbate
1 mM
5 mM
Figure 3. Ascorbate (0-5 mM) demonstrated
significant decreases in clonogenic survival
in MIA PaCa-2 rho+ cells but no changes in
clonogenic survival in MIA PaCa-2 rhoo
cells.
(relative to no ascorbate for each treatment point)
Surviving fraction
1
No ascorbate
Ascorbate 1 mM
0.8
*
0.6
0.4
0.2
0
Control
AntA
*P<0.05 vs ascorbate
(relative to control for each treatment point)
Surviving fraction
1
0.8
0.6
*
0.4
0.2
*
*
0
myx
rot
DNP
AntA
Control
METC
Ascorbate
Ascorbate + METC
*P<0.05 vs ascorbate
(relative to control for each treatment point)
Surviving fraction
1
0.8
0.6
Control
METC
Ascorbate
Ascorbate + METC
*
0.4
0.2
*
*
*
*
0
antA
myx
rot
DNP
(relative to no ascorbate for each treatment point)
Surviving fraction
1
No ascorbate
Ascorbate 2 mM
0.8
0.6
0.4
*
0.2
0
Control
DNP
(relative to no ascorbate for each treatment point)
Surviving fraction
1
0.6
No ascorbate
Ascorbate 2 mM
0.8
*
*
0.4
0.2
0
Control
rotenone
(relative to no ascorbate for each treatment point)
Surviving fraction
1
No ascorbate
ascorbate
0.8
0.6
0.4
*
0.2
0
Control
myxothiazol
Figure 4. The addition of the mitochondrial electron
transport chain (ETC) blocker Antimycin A (AntA) to
ascorbate decreased human pancreatic cancer (MIA
PaCa-2) clonogenic survival, relative to the use of the
AntA alone. MIA PaCa-2 cells were treated for one hour
with and without ascorbate 1 mM for one hour in the
presence of Antimycin A 10 M for 4 hours. MIA PaCa-2
cells were treated with ascorbate are represented as clear
bars per treatment group. Dark bars represent the
surviving fraction of cells treated without ascorbate. P <
0.05 vs. no ascorbate, N=3.
*P < 0.05 vs 0 mM Ascorbate
H2O2 (M)
250
*
200
*
150
100
50
*
0
0 mM
0.1 mM
Ascorbate
5 mM
10 mM
*P < 0.05 vs 0 mM Ascorbate
*
*
60
H2O2 (M)
50
*
40
30
20
10
0
0
20
Time (min.)
40
60
Figure 5. H2O2 generation in cell culture
medium. H2O2 was measured by oxygen
electrode. H2O2 increased as a function of
ascorbate concentration and a function of
time (ascorbate 1 mM).
(normalized to 0 mM for each cell line)
Viability
1
0 mM
5 mM
10 mM
0.75
0.5
*
*
0.25
*
*
* * *
*
* *
0
MiaPaCa-2
AsPC-1
BxPC-3
H6c7
K-ras+
*P<0.05 vs 0 mM for each cell line
Figure 6. Effects of pharmacologic ascorbic acid
concentrations on pancreatic cancer and pancreatic
ductal epithelial cells. All cells were treated with
ascorbate (0, 5, 10 mM) for one hour. Cell
viability determined by MTT assay. MIA PaCa-2,
AsPC-1, BxPC-3 are pancreatic cancer cell lines.
Immortalized pancreatic ductal epithelial cell line,
H6c7 and its derivatives, and H6c7er-Kras (H6c7
cells expressing K-ras oncogene), also received
ascorbate (0, 5, 10 mM) for one hour.
Conclusions
• Pharmacological doses of ascorbate,
achievable in humans when given
intravenously, may have potential for
therapy in pancreatic cancer.
• The ascorbate-induced cytotoxicity in
pancreatic cancer cells may be mediated by
a mitochondrial mechanism.
Animal Protocol
2 x 106 MIA PaCa-2
Pancreatic cancer cells
Day 1
Ascorbate 4 g/kg or
Saline (1M)
q day x 14 days
Day 14
Stop treatment
1000
saline
Ascorbate
800
600
Ascorbate (4 g/kg I.P.every
day for 14 days).
Hypertonic saline (g/kg I.P.
every day for 14 days).
400
200
0
1
5
8
Days
12
15
1
Survival
0.75
Saline (1M)
Ascorbate (4 g/kg)
0.5
0.25
0
1
6
11
16
Days
21
26
31
36
1
Survival
0.75
Saline (1M)
Ascorbate (4 g/kg)
0.5
0.25
0
1
18
Days
36
*P< 0.01 vs control
0.9
(relative to control)
Surviving fraction
1.2
0.6
0.3
*
0
Control
Ascorbate 2
mM
Catalase 100
ug/ml
Catalase +
Ascorbate
PEG-Catalase PEG-Catalase +
200 u/ml
Ascorbate
*P< 0.01 vs control
1
*
*
(relative to control)
Surviving fraction
0.8
0.6
0.4
0.2
*
0
Control
Ascorbate 2 mM
2DG 25 mM
Ascorbate + 2DG
1
control
ascorbate
0.1
0.01
control
2DG
DHEA
BSO