Potential mechanisms of ascorbate-induced cytotoxicity in pancreatic cancer cells

1
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.

2
Abstract Pharmacological concentrations of
ascorbate easily achieved in humans may be
effective in cancer therapeutics (PNAS, 1048749,
2007). We hypothesized that ascorbate
concentrations achievable with intravenous dosing
may be cytotoxic in pancreatic cancer where the
five year survival is lt 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., 655045, 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. 28137416,
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 rhoo 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.
3
Introduction

• Pharmacological concentrations of ascorbate
  easily achieved in humans may be effective in
  cancer therapeutics (PNAS, 1048749, 2007).

4
Predicted plasma Vitamin C concentrations in
healthy persons after oral (top) or
intravenous (bottom) administration of Vitamin C.
Annals of Internal Medicine 140533, 2004
5

• 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 Moertels
  studies were taken as definitive, ascorbate
  treatment was considered useless. However
  Moertels 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 lt 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 lt 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 gt 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 19423, 2000
6

• 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 5743-66, 2007. Cur Probl. Surg
3659-152, 1999.
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Hypothesis

• Ascorbate concentrations achievable with
  intravenous dosing may be cytotoxic in pancreatic
  cancer where the five year survival is lt 3.

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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., 655045, 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. 28137416, 2006), were treated with
  ascorbate and clonogenic survival determined.
• The oxygen electrode method was used to determine
  H2O2 production.

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Mia PaCa-2 Plt0.05 vs 0 mM Ascorbate Arrow
indicates that no colonies were formed when 20
mM ascorbate was given for one hour
Clonogenic Survival (Clonogenic survival relative
to 0 mM Ascorbate)


Ascorbate
10
AsPC-1 Plt0.05 vs 0 mM Ascorbate

Clonogenic Survival (Clonogenic survival relative
to 0 mM Ascorbate)


Ascorbate
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• Figure 1. MIA PaCa-2 and AsPC-1 pancreatic cancer
  cells were treated with ascorbate (0-20 mM) for
  one hour and clonogenic survival determined.
  Ascorbate caused a dose-dependent decrease in
  clonogenic survival in pancreatic cancer cell
  lines.

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A.
C.
D.
B.
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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.
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Mia PaCa-2 rho cells Plt0.05 vs 0 mM Ascorbate

Clonogenic Survival (Clonogenic survival relative
to 0 mM Ascorbate)


Ascorbate
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Mia PaCa-2 rhoo cells Plt0.05 vs 0 mM Ascorbate
Clonogenic Survival (Clonogenic survival relative
to 0 mM Ascorbate)
Ascorbate
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• 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.

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Surviving fraction (relative to no ascorbate for
each treatment point)
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Plt0.05 vs ascorbate

Surviving fraction (relative to control for each
treatment point)


19
Plt0.05 vs ascorbate

Surviving fraction (relative to control for each
treatment point)




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Surviving fraction (relative to no ascorbate for
each treatment point)

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Surviving fraction (relative to no ascorbate for
each treatment point)
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Surviving fraction (relative to no ascorbate for
each treatment point)

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• 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 lt 0.05 vs. no ascorbate,
  N3.

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P lt 0.05 vs 0 mM Ascorbate


H2O2 (?M)

Ascorbate
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P lt 0.05 vs 0 mM Ascorbate



H2O2 (?M)
Time (min.)
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• 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).

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Viability (normalized to 0 mM for each cell line)










Plt0.05 vs 0 mM for each cell line
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• 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.

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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.

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Animal Protocol
Day 1 Ascorbate 4 g/kg or Saline (1M) q day x 14
days
Day 14 Stop treatment
2 x 106 MIA PaCa-2 Pancreatic cancer cells
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Ascorbate (4 g/kg I.P.every day for 14 days).
Hypertonic saline (g/kg I.P. every day for 14
days).
Tumor volume (mm3)
Days
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Survival
Days
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Survival
Days
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Plt 0.01 vs control
Surviving fraction (relative to control)

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Plt 0.01 vs control


Surviving fraction (relative to control)

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