Published OnlineFirst July 20, 2010; DOI: 10.1158/0008-5472.CAN-09-4615
Fructose Induces Pancreatic Cancer Growth
5 mmol/L [1,2-13C2]D-fructose–containing (>99% purity and
99% isotope enrichment for each carbon position; Cambridge
Isotope Labs) media (half unlabeled glucose/fructose, half la-
beled with the 13C tracer) in T75 culture flasks. Where pos-
sible, these studies were also performed in primary
pancreatic tumor and normal pancreas cultures. Briefly, fol-
lowing glucose or fructose treatment, culture medium was
collected and cells were washed twice in PBS, after which
cell pellets were harvested, specific extractions were per-
formed as described below, and mass spectral data were
obtained on a HP5975 inert XL mass selective detector
connected to an HP6890N gas chromatograph. A HP-5 cap-
illary column was used for the glucose, ribose, and lactate
analyses. Lactate was extracted from cell culture media
(0.2 mL) by ethylene chloride after acidification with HCl, de-
rivatized to its propylamine-heptafluorobutyrate ester form,
and applied to the column. The m/z 328 (carbons 1–3 of lac-
tate; chemical ionization) were monitored for the detection
of m1 (recycled lactate through the PC) and m2 (lactate pro-
duced by the Embden-Meyerhof-Parnas pathway) for the
estimation of pentose cycle activity. For analysis of fatty acid
synthesis, palmitate, stearate, cholesterol, and oleate were
extracted after saponification of cell pellets in 30% KOH
and 100% ethanol using petroleum ether. Fatty acids were
then converted to their methylated derivative using 0.5 N
methanolic-HCl, and palmitate, stearate, and oleate were
monitored at m/z 270, m/z 298, and m/z 264, respectively,
with the enrichment of 13C-labeled acetyl units to reflect syn-
thesis, elongation, and desaturation of the new lipid fraction
as determined by mass isotopomer distribution analysis
(MIDA). To compare ribose and deoxyribose synthesis,
RNA ribose was isolated by acid hydrolysis of cellular RNA
after Trizol purification of cell extracts. Total RNA was first
quantified by spectrophotometric determination in triplicate
cultures, and ribose was then derivatized to its aldonitrile
acetate form using hydroxylamine in pyridine with acetic an-
hydride (Supelco) before mass spectral analyses. The ion clus-
ter was monitored around the m/z 256 (carbons 1–5 of ribose;
chemical ionization), m/z 217 (carbons 3–5 of ribose), and m/z
242 (carbons 1–4 of ribose; electron impact ionization) to de-
termine molar enrichment and the positional distribution of
13C in ribose. Ribose molecules labeled with a single 13C atom
on the first carbon position (m1) recovered from RNA were
used to gauge the ribose fraction produced by direct oxida-
tion of glucose or fructose through the glucose-6-phosphate
dehydrogenase (G6PDH) pathway. Ribose molecules labeled
with 13C on the first two carbon positions (m2) were used
to measure the fraction produced by TK. Doubly labeled ri-
bose molecules (m2 and m4) on the fourth and fifth carbon
positions were used to measure molar fraction produced by
triose phosphate isomerase and TK.
Materials and Methods
Cells
Human pancreatic cancer (CaPan-I, CaPan-II, HPAF2,
Aspc1, Panc-1, and MiaPaCa-2) and hepatoblastoma (HepG2)
cell lines were purchased in 2009 and 2010 from the American
Type Culture Collection (ATCC) and used in the described
experiments within 6 months after purchase. Authentication
testing was performed by ATCC and includes (a) certification
that each cell line is negative for Mycoplasma, bacteria, and
fungi contamination; (b) confirmation of species identity and
detection of possible cellular contamination or misidentifica-
tion using Cytochrome c Oxidase subunit I (COI) for interspe-
cies identification and short tandem repeat (STR) analysis
(DNA profiling) for intraspecies identification; and (c) con-
ducting of additional test methods, such as cytogenetic anal-
ysis (G-banding and fluorescence in situ hybridization), flow
cytometry, and immunocytochemistry as well as consistent
refinement of cell growth conditions as well as documenta-
tion systems, ensuring traceability.
The immortalized pancreatic epithelial cell line HPDE6
was kindly provided by Dr. Stephen Pandol (Veteran's Ad-
ministration Medical Center, Los Angeles, CA), and all experi-
ments were performed within five passages (15). For primary
cultures, freshly resected pancreatic tumors or adjacent nor-
mal pancreas tissues were mechanically and enzymatically
(trypsin and DNase) disaggregated, and aliquots of tumor
cells were seeded in six-well plates. Cell viability was con-
firmed using a viability kit (Molecular Probes), and keratin
staining was used to confirm that >98% of the isolated pan-
creatic cancer cells were of epithelial origin. Cell aliquots
were then incubated in standard DMEM in a range of fetal
bovine serum (FBS) concentrations (1–10%) and in a range
of glucose or fructose concentrations for 24 to 72 hours.
In vitro proliferation assays
For CellTiter-Glo (CTG; Promega), fluorescence-activated
cell sorting (FACS), and bromodeoxyuridine (BrdUrd) prolif-
eration assays, 1 × 104 cells were preincubated in standard
medium containing 10% FBS (which contains ∼0.4 mmol/L
glucose) overnight and then plated in a range of glucose- or
fructose-containing standard media in 96-well plates for 12
to 96 hours. For CTG assays, proliferation rates were measured
according to the manufacturer's instructions. For FACS anal-
ysis, treated cells were trypsinized, centrifuged (1,500 rpm ×
2 minutes), washed with PBS, and treated with 20 g/mL RNase
A (Calbiochem). DNA was stained with 100 μg/mL propidium
iodide for 30 minutes at 4°C and protected from light before
analysis with a FACScan (Becton Dickinson). For BrdUrd
uptake assays, BrdUrd (10 μmol/L) was added in the final
12 hours of incubation in the sugars, following which BrdUrd
uptake was quantified.
TK expression
Metabolomic studies
Quantitative reverse transcription-PCR (RT-PCR) was used
to measure TKT, TKTL1, and TKTL2 mRNA levels. Briefly,
RNA was extracted from pancreatic cancer cells (CaPan-I,
CaPan-II, HPAF2, Aspc1, Panc-1, and MiaPaCa-2) using
RNeasy Mini kit (Qiagen). RNA was reverse transcribed
Confluent cultures (75%) of pancreatic cancer (Panc-1 and
MiaPaCa-2), hepatoblastoma (HepG2), and normal pancre-
atic ductal (HPDE6) cells (3 × 106) were incubated for 24
and 72 hours in 5 mmol/L [1,2-13C2]D-glucose–containing or
Cancer Res; 70(15) August 1, 2010
6369
Research.