1. Introduction
Materials and Methods
Discussion
 To investigate if high doses ASC can kill cancer cells such as
HepG2 hepatocarcinoma or SKNSH neuroblastoma cells.
 The observe if the amount of antineoplastic agent, in particular
sorafenib, needed to cause cytotoxic to cancerous cells can
be decreased when taken together with ASC.
 To study the effect of high dose ASC on non-cancerous such as
HEK kidney cells.
Objectives
Conclusions
Results
 This study aimed to assess if high dose ASC has toxic effect on
cancer cells, such as HepG2 hepatocarcinoma or SKNSH
neuroblastoma. Different concentration of ASC and other
compounds were tested for their cytotoxic and non-cytotoxic
effects.
 In SKNSH, ASC at 5 mM caused more cytotoxic effect than
glutathione, or N-Acetyl Cysteine, or Resveratrol. When ASC
were added to 100 M SF, its combined effect showed more a
favorable response than the combined effect of SF+GLU,
SF+NAC, or SF+ RSV.
 The study also examined the cytotoxic effect SF on cells. SF
caused cell death in a dose-dependent manner.
 In HepG2, 5 mM ASC and 100 M SF caused pronounced cell
death.
 This study provides further documentation that 5 mM ASC can
cause cell death when given with lower dose of SF at 3.2 M.
 ASC at a very high dose (100 mM) did not cause cell death in
HEK kidney cells.
• HepG2 hepatocarcinoma, SKNSH neuroblastoma, and HEK 293
kidney cell lines, Eagle’s Minimum Essential Medium (EMEM),
Dulbecco’s Eagle’s Medium (DMEM), fetal bovine serum (FBS)
were obtained from the American Type Culture Collection
(ATCC).
• 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazoliumbromide(MTT),
sulforhodamine B, acetic acid, trypsin/EDTA, tricholoro acetic
acid (TCA), Tris, HCl, isopropanol were obtained from Sigma
Aldrich.
• HepG2 and SKNSH were maintained in a monolayer culture in
EMEM with 100 IU/ml penicillin/streptomycin and 10% FBS.
• HEK cells were maintained in DMEM with similar concentrations
of penicillin/streptomycin and FBS.
• Cytotoxicity Sulforhodamine B Assay is based on method by Sun
et al.,1997.3 Briefly, cells were trypsinized with trypsin/EDTA to
detach the cells. Cells was diluted with culture medium to a
concentration of 2x 105 per ml. 100 l of the cell suspension were
plated into 96 wells plate. After incubation in an incubator
maintained at 37C at 5% CO2 overnight, the medium was
aspirated. The plate were filled with appropriate concentrations of
ASC, SF, or other compound diluted in medium. The plate was
incubated for 72 hrs at 37C. 3 days after the treatment, culture
medium was removed and cells were fixed with 100 l 10% TCA
at 4 C for at least 1 hr. After the TCA was removed from the
plate, cells were washed once with 150 l water followed by
staining with 50 l 0.4% sulforhodamine B dissolved in 1% acetic
acid for 15 minutes. Cells were washed with 100 l of acetic acid
3x to remove unbound stain. Bound protein stain was dissolved
with 150 l of 10 mM Tris-HCl pH7.5. After the cells were
completely dissolved by gentle shaking, the absorbance of the
cells was read at 570 nm using a Microplate Reader Spectramax
M3 (Molecular Device).
• MTT assay is based on method by Mosmann,1983.4 Briefly, cells
were seeded with medium at 2x 105 per ml in which 100 l of
suspension was plated into 96 wells which were then incubated
at 37C for 48 hrs. ASC, SF, or other compounds diluted in
medium were added to the plate, and the plate was incubated for
another 48 hrs. Culture medium was removed and changed with
fresh medium before the addition of 5 mg/ml of 10 l MTT. Cells
were incubated with MTT for 4 hr. at 37 C. Acid-isopropanol
(100 l of 0.04N HCl in isopropanol) was added and mixed to
dissolve the insoluble formazan. The absorbance was read at
570 nm using a Microplate Reader.
 The functional role of ascorbic acid (ASC) in cancer biology is
complex. It was proposed that administration of intravenous (iv)
ASC yields 61-150 fold higher dose than when taken orally.
Proposed mechanism of how high doses of ASC causing cell
death to cancer cells is by the generation of toxic H2O2 and
formation of ASC radicals which were transferred into the
extravascular tissues. 1
 H2O2 exerts cytotoxic effect on normal and cancer cells.
 Sorafenib (SF) is a tyrosine kinases (VEGFR, PDGFR) and Raf
kinases inhibitor. It is used to treat advanced renal cell
carcinoma, unresectable hepatocellular carcinomas and thyroid
cancer. 2
Disclosure
The authors of this poster have no financial conflicts of interest to disclose.
MTT cell viability data
Cytotoxic data
References
EFFECT OF HIGH DOSE ASCORBIC ACID AND SORAFENIB ON
NEUROBLASTOMA, HEPATOCARCINOMA AND KIDNEY CELL LINES
Dr. Lunawati L Bennett,* Ife Babatunde, Anthony Duong, Rene Effoe
*Associate Professor of Pharmaceutical Sciences, Union University School of Pharmacy
1050 Union University Drive, Jackson, TN 38305
llbennett@uu.edu
1. Chen Q, Espey MG, Sun AY, et al. Ascorbate in pharmacologic
concentrations selectively generates ascorbate radical and
hydrogen peroxide in extracellular fluid in vivo. Proc Natl Acad
Sci USA 2007;104:8749-8754.
2. Keating GM, Santoro A. Sorafenib: a review of its use in
advanced hepatocellular carcinoma. Drugs 2009;69(2):223–
240.
3. Sun P, Yue M, Dawson et al. Differential effects of synthetic
nuclear retinoid receptor-selective retinoids on the growth of
human non-small cell lung carcinoma cells. Cancer Research
1997;57 (21):4931-4939.
4. Mosmann, T. Rapid colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxicity assays. J
Immunol Methods 1983;65:55-63.
The graphs illustrates changes in the number of cells before (100%) and after
treatments. The data were based on average of 8 wells (n=8). Each experiment
was run in triplicate. Bars represent the means + standard deviation.
p<0.05 (student t test) compared to non-treated cells.
ASC=ascorbic acid; SF=sorafenib; GLU=glutathione; RSV=resveratrol
NAC=N-acetyl cysteine.
The graphs illustrates changes in the number of cells before (100%) and after
treatments. The data were based on average of 8 wells (n=8). Each experiment
Was run in triplicate. Bars represent the means + standard deviation.
p<0.05 (student t test) compared to non-treated cells.
ASC=ascorbic acid; SF=sorafenib; GLU=glutathione; RSV=resveratrol
NAC=N-acetyl cysteine.
 High dose ASC 5 mM alone or in combination a with lower
concentration of SF showed powerful cytotoxic effects on
cancer cell lines such as HepG2 and SKNSH.
 High dose ASC 5 mM or above didn’t have detrimental effect on
HEK kidney cells.
Acknowledgement: Dr. David Kuhl, Professor of Union University
School of Pharmacy for his thoughtful review and input into the
data analysis and poster development.