1. The Effects of E. coli Strains on Human Colon Cancer Cell Line Caco2
Alterations in the gut microbial composition have been associated with
colorectal cancer (CRC), a leading cause of cancer death in the United
States. However, the precise mechanism by which gut bacteria contribute
to CRC is not clearly defined. In this study we investigated the effects of
bacteria (E. coli) on the Caco2 human colon cancer cell line. Using
antibiotic free media we seeded 2 million Caco2 cells in 6-well plates,
and cultured for 24 hours to allow monolayer formation. Then Caco2
cells were exposed to three different E. coli strains (3BB-001; isolated
from an adenoma patient, K12; non-pathogenic, NC101; which is a
pathogenic murine strain) for 8 hours co-culture. Bacteria were removed
by aspiration of supernatant, followed by two washes with DPBS. The E.
coli exposed vs control Caco2 unexposed cells were maintained for
additional culture for 24, 36, 48 hours on antibiotic media, in order to
calculate the cell viability over time. Viability was determined using
trypan blue; viable and non-viable cells were counted at 56, 68, and 80
hours’ time points. Collected data of the cell viability over the time
period was analyzed. E. coli co-culture with Caco2 demonstrated
significantly increased viability at 56, 68 and 80 hours compared to
control Caco2 cells only. The Caco-2 cells alone had a viability much
lower than when co-cultured with bacteria... Additional studies will
examine the effects of bacteria on Caco2 cell proliferation, apoptosis,
and secretion of inflammatory mediators. In these studies we
demonstrate that Caco2 human cancer cells demonstrate enhanced
growth when exposed to the potential pathogenic bacteria E.Coli 3BB-
001, K12, and NC 101.
PARTNERS Research and Training Program in Cancer Disparities
Abstract
Results
Methodology
Acknowledgements
Conclusions
Colorectal cancer (CRC) is one of the leading
causes of cancer deaths worldwide, and in 2012
CRC accounted for approximately 694,000 deaths
worldwide [3]. The actual cause of colorectal cancer
has not been clearly known; but it has been reported
that the different types and abundance of bacteria
within the gut may contribute to CRC (2). The
mechanism by which gut bacteria contributes to
colorectal carcinogenesis is unclear. We determined
the effects of E. coli on Caco2 human colon cancer
cell line in order to gain a better understanding of
the relationship between bacteria and host cells.
Acknowledgements
Brandon Beaty1, Santosh Dulal2, Amber N. McCoy2, Temitope O. Keku2
1. North Carolina Central University, Durham, NC, USA 2. University of North Carolina at Chapel Hill, School of Medicine, Chapel Hill, NC, USA
B. B. was supported by NCCU-LCCC
Partnership Research grant U54 CA156735
from the NCI/NIH from the National
Cancer Institute.
Additional funding for research project
was provided by NIH R01 CA 136887 and
R01 CA 044684.
Special thanks to Dr. Sarah Andres & Dr.
Kay Lund for providing Caco2 cell lines.
 Co-culture of Caco2 and different E. coli strains 3BB-001, K12 and
NC101 were established.
 The results demonstrate that there are significant differences in
rate of viability at different time points between Caco2 cells co-
cultured with E . coli compared to Caco2 cells only.
 Exposure to bacteria enhanced the rate of viability of the Caco2
cells.
 Bacteria might promote the growth advantage of Caco2 co-cultured
with bacteria compared to the normal Caco2 cells only .
 Future studies will assess the exact effects of bacteria on Caco2 cell
proliferation, apoptosis, and secretion of inflammatory mediators.
Figure 4: Non-viable Caco-2 cells at 36
after bacterial exposure (68 hrs time point)
Figure 3: Caco-2 cells at 36 hours after bacteria exposure
(68 hrs time point)
Methods
0
5
10
15
20
25
30
35
40
Non-viable ViableCellcount(105)
Methodology
Caco2
Co-culture with E .coli for 8 hours
2 x 106 Caco2 +
1 x 106 E. coli K12
Count Viable/non-viable Caco2 cells at 56, 68, and 80 hours
24 hours cultured
in 6 well plates &
formed monolayer
Figure 1: Experimental Design
Figure 2. Viability Curve of Caco2 in the presence/
absence of bacteria
Introduction References
1) Ferlay J, Soerjomataram I, Ervik M, Dikshit R, Eser S, Mathers
C, Rebelo M, Parkin DM, Forman D, Bray F: GLOBOCAN 2012
v1.0: Cancer Incidence and Mortality Worldwide: IARC
CancerBase No. 11 (Internet). In. Lyon, France: International
Agency for Research on Cancer. Available from:
http://globocan.iarc.fr; 2013.
2) Hold GL, Smith M, Grange C et al: Role of the gut microbiota in
inflammatory bowel disease pathogenesis: What have we learnt in
the past 10 years? World journal of gastroenterology : WJG 2014;
20:1192-1210.
3) Siegel R, Ma J, Zou Z et al: Cancer statistics, 2014. CA: a cancer
journal for clinicians 2014; 64:9-29.
2 x 106 per well
E .coli removed
2 x 106 Caco2
only
2 x 106 Caco2 +
1 x 106 E. coli NC101
2 x 106 Caco2 +
1 x 106 E. coli 3BB-001
1 x 106 E. coli per well
E. coli
CFUs/mL
after 8
hours
~ 1 x 108
Gut
Microbiome
(Bacteria)
Adenoma
Genetic &
Environmental
Factors.
CRC
Progression
75
80
85
90
95
100
0 10 20 30 40 50 60 70 80
Caco2 Caco2 + K12
Caco2 + NC101 Caco2 + 3BB
%ViabilityofCaco2
Time (Hours)
Bacteria (E. coli) co-culture
Monolayer
formation
0
1
2
3
4
5
6
7
8
Non-viable
Caco2 Caco2+K12
Cellcount(105)
p value (from T-TEST):
* = < 0.05 – 0.01, ** = < 0.01 – 0.001, *** = < 0.001
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