Chronic Oxaliplatin Resistance Induces Epithelial-to-Mesenchymal
T ransitionin Colorectal Cancer Cell Lines
Anthony D.Yang,1Fan Fan,2E. Ramsay Camp,1George van Buren,1Wenbiao Liu,2Ray Somcio,2
MichaelJ. Gray,2Haiyun Cheng,2Paulo M. Hoff,3and Lee M. Ellis1,2
Purpose: Epithelial-to-mesenchymal transition (EMT) is a process whereby cells acquire
molecular alterations that facilitate cell motility and invasion. In preliminary studies, we
observed that oxaliplatin-resistant (OxR) colorectal cancer (CRC) cells underwent morphologic
changes suggestive of a migratory phenotype, leading us to hypothesize that OxR CRC cells
Experimental Design: The human CRC cell lines KM12L4 and HT29 were exposed to
increasing doses of oxaliplatin to establish stable cell lines resistant to oxaliplatin. Migration
and invasion were assessed by modified Boyden chamber assays. Morphologic and molecular
changes characteristic of EMT were determined by immunofluorescence staining and Western
Results:The OxR cells showed phenotypic changes consistent with EMT: spindle-cell shape,
exhibited an f8- to 15-fold increase in migrating and invading cells, respectively (P < 0.005
for both). Immunofluorescence staining of OxR cells revealed translocation of E-cadherin and
h-cateninfromtheirusualmembrane-boundcomplex tothe cytoplasmandnucleus,respectively.
TheOxRcells alsohaddecreasedexpressionof theepithelialadhesionmolecules E-cadherinand
plakoglobin and an increase in the mesenchymal marker vimentin. The KM12L4 OxR cells
exhibited increased nuclear expression of Snail, an EMT-regulatory transcription factor, whereas
the HT29 OxR cells exhibited an increase in nuclear expression of the EMT-associated
transcription factor nuclear factor nB.
Conclusion: We hypothesize that induction of EMT may contribute to the decreased efficacy
of therapy in chemoresistant CRC, as the tumor cells switch from a proliferative to invasive
phenotype. Further understanding of the mechanisms of chemoresistance in CRC will enable
improvements in chemotherapy for metastatic disease.
Oxaliplatin is a third-generation platinum compound and is
the first platinum-based compound to show efficacy in the
treatment of colorectal cancer (CRC; ref. 1). Its use in
combination with 5-fluorouracil and leucovorin (FOLFOX)
for metastatic CRC has led to response rates >50% and median
survival approaching 2 years (2, 3). FOLFOX has also been
found to be very effective in the adjuvant setting, leading to an
increase in the number of patients who are cured after surgical
resection when compared with the use of 5-fluorouracil and
leucovorin alone (4). Despite these impressive accomplish-
ments, virtually all metastatic CRC eventually become resistant
to oxaliplatin, with a median time to progression of
f8 months (5). Hypotheses on the mechanisms of oxaliplatin
resistance include defects in oxaliplatin uptake, impaired DNA
adduct formation, and increased expression of a copper efflux
Epithelial-to-mesenchymal transition (EMT) is a process
initially observed in embryonic development in which cells
lose epithelial characteristics and gain mesenchymal properties
to increase motility and invasion (10). Previous research
suggests that EMT is also important in tumor progression and
metastasis (10, 11) and is induced by growth factors implicated
in these processes such as hepatocyte growth factor, trans-
forming growth factor h, and epidermal growth factor (12).
In preliminary studies, our laboratory observed that
oxaliplatin-resistant (OxR) CRC cells exhibit an altered
phenotype whereby cells disperse, develop pseudopodia, and
assume a spindle shape, properties associated with the EMT
phenotype. Based on the above observations, we hypothesized
Authors’Affiliations: Departments of1Surgical Oncology,2Cancer Biology, and
3Gastrointestinal Medical Oncology,The University ofTexas M.D. Anderson Cancer
Received1/9/06; revised 4/20/06; accepted 5/4/06.
Grant support: NIHCore Grant CA6672, NIH grantT-32 09599 (A.D.Yang, E.R.
Camp, and G. van Buren), and the Lockton Fund for Pancreatic Cancer Research
(M.J. Grayand L.M. Ellis).
The costs of publicationof this articlewere defrayedinpart by the paymentof page
charges.This article must therefore be hereby marked advertisement in accordance
with18 U.S.C. Section1734 solely toindicatethis fact.
Note: A.D.Yangand F. Fancontributedequally to this work.
Requests for reprints: Lee M. Ellis, Department of Surgical Oncology, Unit 444,
The University ofTexas M.D. Anderson Cancer Center, P.O. Box 301402, Houston,
TX 77230-1402. Phone: 713-792-6926; Fax: 713-792-4689; E-mail: lellis@
F2006 American Associationfor Cancer Research.
www.aacrjournals.orgClin Cancer Res 2006;12(14) July15, 20064147
that oxaliplatin resistance leads to an EMT phenotype,
including its characteristic molecular alterations. To test this
hypothesis, we assessed OxR cells derived from two human
CRC cell lines for gross morphologic, immunohistochemical,
and molecular changes consistent with EMT.
Materials and Methods
Cell lines and culture conditions.
was obtained from I.J. Fidler, D.V.M., Ph.D. (The University of Texas
M.D. Anderson Cancer Center, Houston, TX). The human CRC cell line
HT29 was obtained from the American Type Culture Collection
(Manassas, VA). Cell lines were cultured in MEM supplemented with
10% fetal bovine serum (FBS), penicillin-streptomycin, vitamins,
sodium pyruvate, L-glutamine, nonessential amino acids (Life Tech-
nologies, Grand Island, NY), and HEPES buffer (MP Biomedicals,
Irvine, CA) at 37jC in 5% CO2and 95% air. Cells were confirmed to
be free of Mycoplasma using a Mycoplasma Detection Kit (American
Type Culture Collection). In vitro experiments were done at 50% to
70% cell confluence. Results from all studies were confirmed in at least
three independent experiments.
Drugs and antibodies.Oxaliplatin (Sanofi-Synthelabo, New York,
NY) was purchased from the pharmacy at M.D. Anderson Cancer
Center. Antibodies used for immunofluorescence staining and Western
blot analyses were as follows: mouse anti-E-cadherin (Zymed Labora-
tories, Carlsbad, CA), mouse anti-plakoglobin, mouse anti-vimentin,
mouse anti-smooth muscle actin, mouse anti-fibronectin (Chemicon
International, Temecula, CA), mouse anti-N-cadherin, rabbit anti-h-
catenin (Upstate Chemical, Temecula, CA), rabbit anti-Snail, rabbit
anti-Twist, goat anti-Slug, rabbit anti–nuclear factor nB (Santa Cruz
Biotechnology, Santa Cruz, CA), rabbit anti-actin, mouse anti-vinculin
The human CRC cell line KM12L4
(Sigma-Aldrich, St. Louis, MO), and rabbit anti-lamin B1 (Active Motif,
Development of OxR CRC cell lines.
chronically resistant to oxaliplatin, KM12L4 and HT29 cells were
exposed to an initial oxaliplatin concentration of 0.1 Amol/L in MEM
plus 10% FBS. The surviving population of cells was grown to 80%
confluence and passaged twice over 9 days to ensure viability. The
concentration of oxaliplatin the surviving population was exposed to
was then sequentially increased in the same manner to 0.5 Amol/L
(15 days), 1.0 Amol/L (30 days), and finally to the clinically relevant
plasma concentration of 2 Amol/L. For all in vitro studies, each OxR cell
line was used at no higher than 15 passages from creation. To confirm
that the OxR cell lines would remain resistant in vivo, 2 million cells
were injected s.c. into athymic nude mice as previously described (13).
Tumors were harvested after f30 days and tumor cells were replated
and exposed to 2 Amol/L oxaliplatin.
Morphologic analysis.Cells were grown to 70% confluence in
MEM plus 10% FBS (parental cell lines) or MEM plus 10% FBS
plus 2 Amol/L oxaliplatin (OxR cell lines) and visualized at ?20
magnification with a Nikon light microscope (Nikon, Inc., Melville,
NY) with digital photographic capability. The digital images of the
OxR cells and parental cells were compared for morphologic
characteristics consistent with EMT [i.e., loss of polarity (spindle-
shaped cells), increase in intercellular separation, and appearance
of pseudopodia]. Blinded observers classified the images with
regard to presence or absence of morphologic changes consistent
Migration and invasion assays.
assessed with Boyden chambers or modified Boyden chambers
according to the protocol of the manufacturer (Becton Dickinson
Labware, Bedford, MA). Briefly, 100,000 KM12L4 parental and OxR
cells in MEM plus 1% FBS with or without 2 Amol/L oxaliplatin were
placed on each 8.0-Am pore size membrane insert in 24-well plates.
MEM plus 10% FBS, with or without 2 Amol/L oxaliplatin, was placed
To create stable CRC cell lines
Cell migration and invasion were
Fig.1. Acquisition of oxaliplatin resistance induces morphologic
changes consistent with EMT in CRC cells. KM12L4 (A) and
HT29 (B) parental and OxRcells were assessed for morphologic
changes consistent with EMT. Spindle-shaped cells with loss of
polarity (redarrows), increasedintercellular separation (green
arrows), and pseudopodia (white arrows) were notedin the OxR
cells but not in parental cells from both celllines.
www.aacrjournals.orgClin Cancer Res 2006;12(14) July15, 20064148
in the bottom wells as chemoattractants. After 24 hours, cells that did
not migrate were removed from the top side of the inserts with a
cotton swab. Cells that had migrated to the underside of the inserts
were stained with Diff-Quik (Harleco, Gibbstown, NJ) and the cells
on each insert were counted at ?100 magnification. The invasion
assay was done in a similar fashion except the 8.0-Am pore size
membrane inserts were coated with Matrigel. Results were expressed as
mean F SE.
and OxR cells were grown on poly-L-lysine–coated glass coverslips
(BD Biosciences, San Jose, CA) to 40% to 50% confluence; after
48 hours of incubation, the cells were fixed with cold acetone for
1 hour and permeabilized in 0.5% Triton X-100 (Sigma-Aldrich) for
10 minutes. The cells were then blocked with normal horse and goat
serum in PBS. Cells were incubated with primary antibodies (anti-
E-cadherin or anti-h-catenin) overnight at 4jC. The following
morning, the slides were washed with PBS and incubated with the
appropriate FITC-conjugated secondary antibody for 1 hour. The cells
were then washed and incubated with Hoechst 33342 (Invitrogen
Corp., Carlsbad, CA) for nuclear staining, washed, and mounted with
propyl gallate under glass coverslips. The slides were visualized for
immunofluorescence with a laser scanning Olympus microscope
(Olympus America, Inc., Melville, NY).
KM12L4 and HT29 parental
Western blot analysis.
parental and OxR cells were plated and grown to 70% to 80%
confluence. Whole-cell protein was isolated using radioimmunopreci-
pitation assay B protein lysis buffer as previously described (14).
Nuclear protein was extracted with a commercially available kit
(Active Motif). The isolated protein was quantified by a commercially
available modified Bradford assay (Bio-Rad Laboratories, Hercules,
CA). Western blot protein samples were prepared by boiling the
isolated protein with denaturing sample buffer. The protein was then
separated by SDS-PAGE on a 10% polyacrylamide gel and transferred
to a polyvinylidene difluoride membrane (Millipore Corp., Billerica,
MA). The membranes were blocked with 5% nonfat dry milk in TBS
and 0.1% Tween 20 for 1 hour and probed with the appropriate
primary antibody overnight at 4jC. The next morning, the membranes
were washed and incubated with the appropriate horseradish
peroxidase–conjugated secondary antibody (Amersham Biosciences,
Piscataway, NJ) for 1 hour at room temperature. The membranes were
then washed and protein bands visualized by using a commercially
available enhanced chemiluminescence kit (Amersham Biosciences).
To verify the accuracy of whole-cell lysate and nuclear extract protein
loading, membranes were incubated in stripping solution for 30
minutes at 65jC, washed, and reprobed with h-actin, vinculin, or
lamin-B1 antibody as a loading control.
assay. KM12L4 and HT29 parental and OxR cells were plated in
96-well plates at 4,000 per well with MEM plus 10% FBS and
incubated for 24, 48, or 72 hours. At the end of the incubation,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT;
Sigma-Aldrich) was added to achieve a final concentration of
0.5 mg/mL and the cells were incubated for another 60 minutes.
Medium and MTT were then removed, DMSO was added for 1 minute
to induce cell lysis, and absorption was read at 570 nm.
Flow cytometry and cell cycle analysis.
and OxR cells were grown to 70% to 80% confluence in MEM plus
10% FBS (parental cell lines) or MEM plus 10% FBS plus 2 Amol/L
oxaliplatin (OxR cell lines) over 48 hours. After trypsinization, cells
were washed in PBS and fixed in 70% ethanol at 4jC for 2 hours.
DNA staining was done with 10 mg propidium iodide/mL PBS and
2.5 Ag DNase-free RNase (Roche Diagnostics)/mL PBS for at least
30 minutes before flow cytometry in a Coulter EPICS XL flow
cytometer (Beckman Coulter, Inc., Fullerton, CA). Cell cycle profiles
were generated from flow cytometry analysis with MultiCycle software
(Phoenix Flow Systems, San Diego, CA).
Statistical analysis.Student’s t test was used in all statistical
analyses of MTT, invasion, and migration assay results using InStat
Statistical Software version 2.03 (GraphPad Software, San Diego, CA).
Statistical significance was defined as two-tailed P V 0.05.
For protein extraction, KM12L4 and HT29
KM12L4 and HT29 parental
Acquisition of oxaliplatin resistance induces morphologic
changes consistent with EMT in CRC cells. We first noted that
cells from the human KM12L4 and HT29 CRC cell lines that
had acquired resistance to oxaliplatin at the clinically relevant
concentration of 2 Amol/L had a markedly different light-
microscopic appearance from cells of the parental cell lines. The
phenotypic changes observed in OxR cells included loss of cell
polarity causing a spindle-cell morphology, increased intercel-
lular separation signifying loss of intercellular adhesion, and
increased formation of pseudopodia (Fig. 1). These changes are
typical of cells with a mesenchymal phenotype.
OxR CRC cells have increased migratory and invasive
capacity. Boyden chamber assays were done to compare the
migratory capability of KM12L4 OxR and parental cells. At
Fig. 2. OxRCRC cells have increased migratory andinvasive capacity. Boyden
chamber and modified Boyden chamber assays were done to compare the
migratory andinvasive capabilities of KM12L4 OxRand parental cells. A, at
48 hours, the OxRcells showed an f7.5-foldincrease in the number of cells
migratingthroughthe collageninsert. B, alsoat 48 hours,the OxRcells exhibitedan
f15-foldincrease in the number of cells invading through the Matrigel-coated
collageninsert. HPF, high-power field; Ox, oxaliplatin.
Oxaliplatin Resistance Induces EMTin Colorectal Cancer
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48 hours, the OxR cells showed an f7.5-fold increase in the
number of cells migrating through the collagen membrane
(P < 0.001; Fig. 2A). The capacity of OxR cells to invade
through a Matrigel-coated membrane was even greater, with an
f15-fold increase in the number of invading OxR cells
compared with parental cells (P < 0.005; Fig. 2B). The presence
or absence of 2 Amol/L oxaliplatin in the cell culture medium
did not significantly affect the migratory or invasive capacity of
the OxR cells.
OxR CRC cells exhibit changes in the localization of cellular
E-cadherin and b-catenin. One of the hallmarks of EMT is the
breakdown of the cytoplasmic cell adhesion complex (15),
which causes a change in the localization of E-cadherin and
h-catenin from their usual membrane-bound site. The loss of
E-cadherin from the membrane facilitates a loss of homotypic
epithelial cell adhesion and releases h-catenin from its cell-
surface location. The subsequent translocation of h-catenin to
the nucleus allows it to bind to other proteins as part of a
transcriptional complex that mediates other cellular alterations
associated with EMT (16). To investigate the possibility that
oxaliplatin resistance leads to changes in the cellular localiza-
tion of E-cadherin and h-catenin, immunofluorescence staining
was done on KM12L4 (Fig. 3A) and HT29 (Fig. 3B) OxR and
parental cells. In both cell lines, the OxR cells exhibited a
change in E-cadherin from an organized, membrane-bound
structure to a disorganized state in which it was noted to be
dispersed throughout the cytoplasm. Furthermore, h-catenin
was observed to translocate from the usual membrane-bound
site observed in the parental cells to the nucleus in both OxR
OxR CRC cells exhibit molecular changes consistent with
EMT. To determine if the acquisition of oxaliplatin resistance
induced the specific molecular changes consistent with EMT,
Western blotting was done on cell lysates and nuclear extracts
from the KM12L4 OxR and parental cells (Fig. 4A). Expression
of the epithelial adhesion molecules E-cadherin and plako-
globin was decreased in the OxR cells compared with the
parental cells. A concurrent marked increase in the expression
of the mesenchymal marker vimentin was also observed.
There was no change in expression of the mesenchymal
markers N-cadherin, a-smooth muscle actin, and fibronectin
(data not shown). Furthermore, increased nuclear expression
of the EMT-related transcription factor Snail was observed in
OxR KM12L4 cells compared with the parental cells (15, 17).
No significant changes were observed in nuclear levels of the
EMT-related transcription factors Slug and Twist (also known
as Snail-2 and Snail-3, respectively). Similar results were noted
when validation studies were done in HT29 OxR and parental
cells for the epithelial markers E-cadherin and plakoglobin. In
contrast to the KM12L4 OxR cells, no change in expression of
Fig. 3. OxRCRC cells exhibit changes inlocalization of cellular
EMT markers. Immunofluorescence staining for E-cadherin and
h-catenin was done on KM12L4 (A) and HT29 (B) parental and
OxRcells. OxRcells from both celllines showed changes in
localization of E-cadherin and h-catenin from their usual cell
membrane-associated site. OxRcells exhibited E-cadherinin a
disorganized cytoplasmic location and h-catenin was noted to
translocate to thenucleus.
www.aacrjournals.orgClin Cancer Res 2006;12(14) July15, 2006 4150
mesenchymal markers (data not shown) or nuclear expression
of Snail, Slug, and Twist was observed in the HT29 OxR cells
when compared with the parental cells (Fig. 4B). However, in
the HT29 OxR cells, nuclear expression of the transcription
factor nuclear factor nB was found to be increased (Fig. 4B).
The expression level of h-catenin was not changed in either
the KM12L4 or HT29 OxR cell line when compared with the
parental cell lines in whole-cell protein extracts, cytoplasmic
protein extracts, or nuclear protein extracts (data not shown).
OxR CRC cells exhibit reduced proliferation rates. The
KM12L4 (Fig. 5A) and HT29 (Fig. 5B) parental and OxR cell
numbers were compared at different time points after plating
by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) assay. At 48 and 72 hours, cell counts increased
in all groups but both the KM12L4 and HT29 OxR cell lines
exhibited reduced cell counts compared with the parental cell
lines (P < 0.005 for both comparisons). Cell cycle analysis
showed an increase in the percentage of cells in G2and no
change in the percentage of cells undergoing apoptosis (sub-G0;
Table 1). This suggests that the finding of decreased cell number
in the MTT assay can be attributed to decreased proliferation of
the OxR cells.
The last decade has witnessed major innovations in the
treatment of metastatic CRC. The median survival for affected
patients has increased steadily and is now crossing the 2-year
barrier (18). The advent of oxaliplatin played a major role in this
disease and it is now part of the most commonly used regimens
both in the metastatic as well as in the adjuvant setting.
However, it is far from being the perfect treatment. Despite the
oxaliplatin activity, the average responding patient can expect to
develop resistance f6 to 8 months after the treatment was
initiated. Given enough time, the development of resistance is
uniform, and rare patients with metastatic disease, if any, are
cured without surgery. Cellular levels of glutathione and ERCC-
1 seem to be associated with inherent resistance to oxaliplatin
and may play a role in the acquired resistance as well (7).
Decreased cellular uptake and adduct formation have been
shown but the mechanisms of development of resistance during
treatment are still poorly understood (8).
We showed in this series of experiments that acquisition
of oxaliplatin resistance by CRC cells leads to morphologic
and molecular alterations consistent with a change to a
mesenchymal-like phenotype. Furthermore, we showed that
OxR CRC cell lines had changes in the cellular localization
of two major factors associated with EMT, E-cadherin and
h-catenin. Although expression of E-cadherin was decreased in
both OxR cell lines, we did not observe changes in h-catenin
protein expression levels in whole-cell, cytoplasmic, or nuclear
extracts. This lack of change in h-catenin expression could
be due to high constitutive expression of h-catenin in these
cell lines secondary to APC mutation or it may reflect the
Fig. 4. OxRCRC cells exhibit molecular changes consistent with
EMT. A, celllysates and nuclear extracts from KM12L4 parental and
OxRcells were subjected toWestern blotting. Expression of the
epithelial cellular adhesion molecules E-cadherin and plakoglobin
was decreasedin OxRcells compared with parental cells (top left).
A markedincrease in the expression of the mesenchymal marker
vimentin was concurrently observed (top right).There was also
increased expression of the EMT-related transcription factor Snailin
nuclear extracts of OxRcells compared with parental cells (bottom).
No change in expression of the EMT-related transcription factors
Slug orTwist was noted. B, similar results were noted when
validation studies were done in the HT29 parental and OxRcells
(left) except that no up-regulation of mesenchymal markers was
noted and no changes were notedin Snail, Slug, orTwist. However,
nuclear expression of another transcription factor implicatedin the
induction of EMT, nuclear factor nB (NF-jB), was observedin the
HT29 OxRcellline (right).
Oxaliplatin Resistance Induces EMTin Colorectal Cancer
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fact that this may be a relocation phenomenon that is not
accompanied by changes in h-catenin protein levels. Of note,
although we discovered molecular evidence that the EMT
changes in the KM12L4-OxR cell line were associated with
an increase in the nuclear expression of the transcription
factor Snail, we were not able to detect changes in Snail, Slug,
or Twist in the HT29 OxR cell line. However, nuclear
expression of the transcription factor nuclear factor nB, which
has been shown to be associated with EMT in a mammary
carcinoma model (19), was increased in the HT29 OxR cell
line only. Given that we were unable to observe an increase in
any mesenchymal markers in the HT29 OxR cell line, we
postulate that the HT29 OxR cells may be undergoing an
incomplete change to a mesenchymal-like phenotype rather
than the full EMT observed in the KM12-OxR cells. This could
be explained by the different signaling pathways (Snail versus
nuclear factor nB) inducing the phenotypic change in the two
OxR cell lines. Finally, we also showed that the OxR cells
undergoing EMT had increased migratory and invasive capa-
bilities. This finding of increased aggressiveness was consistent
in two OxR CRC cell lines.
Recent research has implicated EMT in cancer progression by
noting that epithelial-derived tumor cells can switch their
phenotype to a more primitive mesenchymal phenotype that
facilitates motility and invasion (10). Several studies have
examined the possible role of EMT in CRC progression
(20, 21). Although two studies have shown that loss of
E-cadherin-mediated adhesion decreases chemoresistance (22,
23), to our knowledge, no studies have described EMT with
loss of E-cadherin-mediated adhesion occurring in cells that
have acquired chemoresistance. We believe that our finding
that OxR CRC cells undergo epithelial to mesenchymal or
mesenchymal-like transition reflects an important process by
which cancer cells may potentially acquire chemoresistance.
We further hypothesize that OxR cells may switch their
‘‘molecular machinery’’ from a proliferative, epithelial pheno-
type to a more invasive and migratory mode. Because
proliferation is required for oxaliplatin-induced chemosensi-
tivity, the decrease in proliferation of OxR cells may be one
means whereby resistant cells can escape the effects of
In conclusion, this is, to our knowledge, the first description
of chemoresistance-induced EMT, and we postulate that EMT
induced by acquisition of oxaliplatin resistance could be a
possible survival mechanism for chemoresistant CRC cells. If
our hypothesis is true, blocking or reversing EMT changes may
cause chemoresistant cells to revert to chemosensitive cells.
Furthermore, it is yet to be determined if these findings in
OxR cells are applicable in cells that develop resistance to
other chemotherapeutic drugs. We believe that induction of
EMT in chemoresistant CRC represents a new potentially
exciting area of research into the mechanism of CRC
progression and may eventually be of benefit to patients with
advanced, chemoresistant CRC patients who currently do not
have many effective treatment options.
We thank Melissa G. Burkett from the Department of Scientific Publications
and Rita Hernandez from the Department of Surgical Oncology for editorial
Fig. 5. OxRCRC cells have reduced cellnumber.The cellnumber of KM12L4 (A)
and HT29 (B) parentaland OxRcells were comparedby MTTassay. At 24,48, and
72 hours, both the KM12L4 and HT29 OxRcells exhibited reduced cellnumber
compared with the respective parental cells.
Table1. Cell cycle analysis of KM12L4 and HT29 OxR cells
48 hoursSub-G0(%)G1(%) S (%)G2(%)
www.aacrjournals.orgClin Cancer Res 2006;12(14) July15, 20064152
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