# Deoxycholate, an Endogenous Cytotoxin/Genotoxin, Induces   the Autophagic Stress-Survival Pathway: Implications for Colon Carcinogenesis

Authors: Claire M. Payne; Cheray Crowley-Skillicorn; Hana Holubec; Katerina Dvorak; Carol Bernstein; Mary Pat Moyer; Harinder Garewal; Harris Bernstein
Journal: Journal of Toxicology
 (2009)

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## Abstract

We report that deoxycholate (DOC), a hydrophobic bile acid associated with a high-fat diet, activates the autophagic pathway in non-cancer colon epithelial cells (NCM-460), and that this activation contributes to cell survival.  The DOC-induced increase in autophagy was documented by an increase in autophagic vacuoles (detected using transmission electron microscopy, increased levels of LC3-I and LC3-II (western blotting), an increase in acidic vesicles (fluorescence spectroscopy of monodansycadaverine and lysotracker red probes), and increased expression of the autophagic protein, beclin-1 (immunohistochemistry/western blotting).  The DOC-induced increase in beclin-1 expression was ROS-dependent.  Rapamycin (activator of autophagy) pre-treatment of NCM-460 cells significantly (P<.05) decreased, and 3-MA (inhibitor of autophagy) significantly (P<.05) increased the cell loss caused by DOC treatment, alone. Rapamycin pre-treatment of the apoptosis-resistant colon cancer cell line, HCT-116RC (developed in our laboratory), resulted in a significant decrease in DOC-induced cell death.  Bafilomycin A1 and hydroxychloroquine (inhibitors of the autophagic process) increased the DOC-induced percentage of apoptotic cells in HCT-116RC cells.  It was concluded that the activation of autophagy by DOC has important implications for colon carcinogenesis and for the treatment of colon cancer in conjunction with commonly used chemotherapeutic agents.


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## Body

## 1. Introduction

Colon  cancer is  the
second leading cause of cancer deaths among men and women combined in the United States, Australia, and Europe and is a major problem in many
other countries. Approximately 50% of
colorectal cancers are attributed to dietary factors [1]. A typical Western diet, high in fat and low
in fiber, has been shown to contribute to the development of colon cancer in
epidemiologic and animal studies [2]. Bile acids/salts, present in high concentration in the feces of patients
on a high fat/low fiber diet [3], have been associated with colon cancer risk
[4]. The most common bile acid present
in the human feces is deoxycholic acid (DOC) [5], a hydrophobic bile acid. DOC is a promoter of colon cancer [2], and
also a genotoxic carcinogen [6–8], and may be responsible for initiating
gastrointestinal cancers (reviewed by Bernstein et al. [9]). However, the mechanism by which hydrophobic
bile acids act in progression to colon cancer is unclear.Hydrophobic bile
acids are known inducers of at least five stress-response pathways in
gastrointestinal cells, including ER stress [10], oxidative stress [6, 11–13],
nitrosative stress [14, 15], mitochondrial stress [10–13, 16], and DNA damage
[6, 17–19]. Some of these bile acid-induced
cellular stresses may ultimately lead to cell death by mechanisms that include
both apoptosis [20, 21] and necrosis [16]. Hydrophobic bile acids also promote colon cancer. In addition, they may act as carcinogens
[6, 7] and/or select for outgrowth of clones of mutant cells resistant to bile
acid-induced cell death. One of the cell
survival pathways activated in response to bile acid exposure is the NF-κB stress-response pathway [11, 20, 22, 23]. Persistent activation of NF-κB causes cells to become apoptosis-resistant,
and such cells tend to acquire mutations, some of which may contribute to colon
cancer.Another
important cell survival pathway is autophagy. Autophagy (Greek for “the eating of oneself”) is an evolutionarily
conserved lysosomal pathway that allows eukaryotic cells (yeast to mammals) to
survive under nutrient starvation conditions [24–26]. Macroautophagy (herein referred to as
autophagy) involves the bulk lysosomal turnover of long-lived proteins, protein
aggregates, and organelles such as damaged mitochondria [27], damaged
endoplasmic reticulum (ER) [28], and ribosomes [29]. The autophagic process occurs in several
stages that begin with the formation of a crescent-shaped isolation membrane
(phagophore) that sequesters organelles, matures into an autophagosome that surrounds
the organelle, followed by the fusion of the autophagosome with a lysosome to
form an autophagolysosome [30, 31]. Hydrolytic enzymes within the acid pH of the interior of the
autophagolysosome, then act to degrade macromolecules, thereby providing
nutrients for the survival of the eukaryotic cell [26]. An analysis of this morphologic process at
the molecular level reveals a complex series of biochemical events involving
the products of numerous autophagy-related genes [24, 26, 32–35]. The autophagy pathway is becoming
increasingly recognized as an important mechanism of tumor cell survival and
drug resistance in cancer chemotherapy [36–38]. A recent study indicated that autophagy is activated in colorectal cancer
cells and contributes to tolerance to nutrient deprivation [39]. We now show, for the first time, that
deoxycholate (DOC) activates the autophagic pathway in noncancer colon
epithelial cells (NCM-460), and that this activation contributes to cell survival. We also show that the constitutive activation
of autophagy contributes to the survival of apoptosis-resistant colon cancer
cells (HCT-116RC) that were developed in our laboratory by repeated exposure to
increasing concentrations of DOC [40].The
present findings, coupled with findings from our in vivo animal model of deoxycholate-induced colonic inflammation
[17] and from our in vitro apoptosis-resistant colon cancer cell lines [40], implicate autophagy in colon
carcinogenesis and suggest an additional mechanism by which hydrophobic bile
acids contribute to colon carcinogenesis. These studies may aid in the identification of potential biomarkers of
the autophagy pathway in the nonneoplastic colonic mucosa of patients at risk
for colon cancer. In addition, combining
inhibitors of autophagy with chemotherapeutic agents commonly used to treat
colon cancer may lead to an improved clinical  outcome.


## 2. Materials and Methods

### 2.1. Chemicals

Sodium deoxycholate (DOC), 3-methyladenine
(3-MA), rapamycin (Rapa) (derived from Streptomyces hygroscopicus), CuDIPS
[copper(II) 3,5-diisopropylsalicylate hydrate), and catalase (10 000–40 000 U/mg
protein) were obtained from Sigma-Aldrich (St. Louis, Mo, USA). Bafilomycin A1, MnTBAP [Mn (III)
tetrakis (4-benzoic acid) porphyrin chloride], HBED [N,N′-Di-(2-hydroxybenzyl)ethylenediamine-N,N′-diacetic
acid)], pepstatin A, and E-64d were from BIOMOL Research Laboratories (Plymouth
Meeting, Pa, USA), hydroxychloroquine sulfate (HCS) was from Acros Organics (Morris
Plains, NJ), and trypan blue was from Gibco BRL Life Technologies (Grand Island,
NY, USA). The concentrations used to modulate
autophagy and cell death in the cell culture experiments are provided
below. In all instances, the specific
concentrations that were reported in the literature were tested for their
effect on the viability of NCM-460 and HCT-116RC cells used in the present
study. In some instances dose-response
curves that assess the concentration of the drug and its effect on cell
viability were performed to ensure that the concentration of the chemical itself
was not too toxic to be used in the proposed experiments. This was especially important for 3-MA, which
is used at a wide range of millimolar concentrations in published reports. The three antioxidants (HBED, MnTBAP, CuDIPS)
used to evaluate the DOC-induced increase in beclin-1 expression were used at
the same concentration (100 μM)
so that direct comparisons can be made. The concentration chosen was well within the range of concentrations
reported in the literature for use with cultured cells (see cited articles
below), and this concentration did not induce any cytotoxicity in the sensitive
NCM-460 cells. CuDIPS: 100 μM
[15]; Bafilomycin A1: 1 nM [41, 42]; Catalase: 1000 U/mg;
E-64d: 10 μg/mL [43]; 3-MA: 4 mM; HBED: 100 μM [44, 45]; HCS: 10 μM [46]; MnTBAP: 100 μM [47]; Pepstatin A: 10 μg/mL [43]; Rapa: 100 μM.


### 2.2. Cell Lines and Tissue Culture Conditions

The NCM-460 colon
cell line was obtained from INCELL Corporation, LLC (San Antonio, Tex, USA)
and cultured in M3:10TM medium to insure that it maintains its characteristic
features. This cell line was not
obtained from a colon cancer and is considered to be a noncancerous colonic
epithelial cell line. HCT-116RC is a
stable apoptosis-resistant colon cancer cell line that was developed in our
laboratory after persistent exposure to increasing concentrations of NaDOC
[40]. This cell line was maintained in
DMEM media supplemented with 10% heat-inactivated fetal calf serum (Omega Scientific,
Tarzana, Calif, USA),
1% MEM nonessential amino acids, 100 μg/mL
streptomycin, 100 U/mL penicillin, and 3.44 mg/mL L-glutamine. Media components were from Gibco BRL Life
Technologies (Grand Island,
NY, USA). All treatments utilizing
NCM-460 cells were performed with the cells in approximate mid-logarithmic
phase to avoid inconsistencies in cellular responses based on growth phase, as
previously described [48]. Starvation
experiments were performed by incubation cells in Hank’s Balanced Salt Solution
(HBSS), as previously described for colon epithelial cells [49]. HBSS was obtained from Gibco/Invitrogen Corp., Carlsbad, Calif, USA.


### 2.3. Assessment of Vacuolar Acidification

NCM460 cells were plated on a fibronectin-coated Costar (Fisher Scientific, Pittsburg, Pa, USA)
96 well black plate at 2 × 105 cells/mL. Cells were treated with 0.4 mM DOC for 30 minutes and 1 hour. After
treatments, cells were spun in an Allegra 6R Centrifuge (Beckman Coulter, Fullerton, Calif, USA)
at 1000 rpm for 5 minutes and the fluorescent dyes added as described below.
Triplicate wells were plated for each experimental protocol, and the intensity
values were normalized for each well using a nucleic acid stain. Mean fold
changes in intensity values (DOC versus control untreated cells) were
statistically compared using Student’s t-test.


#### 2.3.1. Monodansylcadaverine (MDC)

MDC (Fluka-Sigma-Aldrich, St. Louis, Mo, USA) was used at a final concentration of 200μM, and cells were incubated for 30 minutes at 37°C. MDC was removed and Sytox Green (Molecular
Probes/Invitrogen, Carlsbad,
Calif, USA) was added at a final
concentration of 2.5 μM;
cells were incubated for 20 minutes at room temperature. Fluorescence was assessed immediately using
an Optima FLUOstar plate Reader (BMG Labtech, Durham, NC, USA). MDC (excitation 335 nm; emission 508 nm) and
Sytox Green (excitation 504 nm; emission 523 nm) fluorescence were assessed
using appropriate filters. All
fluorescence values were normalized to the amount of cells in individual wells
by obtaining the ratio of MDC fluorescence (assessment of acid vesicles) to
Sytox Green fluorescence (nucleic acid stain).


#### 2.3.2. Lysotracker Red

LysoTracker Red (Molecular Probes/Invitrogen, Carlsbad, Calif, USA) was added to the cells at a final concentration of
100 nM and incubated for 30 minutes at 37°C. Lysotracker Red was removed and Hoechst # 33342 dye (Molecular
Probes/Invitrogen, Carlsbad,
Calif, USA), made up in tissue culture
media at a final concentration of 10μg/mL,
was added to the cells and incubated for 10 minutes at 37°C. Cells were spun at 1000 rpm for 10 minutes,
the supernatant removed, and the pelleted cells were fixed with 4% formaldehyde
in PBS for 20 minutes at room temperature. Fluorescence at 30 minutes and 1 hour was assessed immediately using an
Optima FLUOstar plate Reader (BMG Labtech, Durham, NC, USA). LysoTracker Red (excitation 577 nm; emission
590 nm) and Hoechst (excitation 350 nm; emission 461 nm) fluorescence were
assessed using appropriate filters. All
fluorescence values were normalized to the amount of cells in individual wells
by obtaining the ratio of LysoTracker Red fluorescence (assessment of acid
vesicles) to Hoechst fluorescence (nucleic acid stain).


### 2.4. Western Blot Analysis

Cells
were grown in 20 × 100 mm Falcon polystyrene tissue culture dishes (Fisher
Scientific, Pittsburgh, Pa, USA). Cultures treated with DOC or incubated in control media were disrupted
in lysis buffer (50 mM Tris pH 8, 5 mM EDTA, 150 mM NaCl, 0.5% NP-40) supplemented
with 1 mM phenylmethylsulfonyl fluoride (PMSF), leupeptin (1μg/mL), and aprotinin (0.01 U/mL). Cell lysates were prepared at a concentration
of 2 μg/μL of protein, and 10 μg of protein were added to each well of a 15%
Criterion Tris-HCl gel (Biorad, Hercules, Calif, USA) for
size fractionation by electrophoresis. The proteins were blotted onto
Immobilon-P PVDF transfer membrane (Millipore, Bedford, Mass, USA).
The membranes were incubated with a mouse antibeclin monoclonal antibody (BD
Transduction Laboratories, San Diego, Calif, USA) at a dilution of 1:1000 or rabbit anti-LC3B
polyclonal antibody (Cell Signaling Technology, Inc., Boston, Mass, USA) at a dilution of 1:500. The membranes were then incubated with goat antimouse or goat
antirabbit secondary antibodies conjugated to horseradish peroxidase (Pierce, Rockford, Ill, USA). Antibody complexes were detected using the
SuperSignal West Pico chemiluminescence detection system (Pierce, Rockford, Ill, USA). Finally, the membranes were stained for 20
minutes with Brilliant Blue G dye (Sigma-Aldrich, St. Louis, Mo, USA)
to confirm equal protein loading. We
have chosen to use the staining of the membranes with Brilliant Blue G dye
rather than a specific protein as a loading control, since the former looks at
numerous bands, whereas the latter can be misleading. This is based on work published from our
laboratory using GAPDH and G3PD [50], and those of others who screened 22
housekeeping genes [51] and found a large number to be modulated by various
experimental conditions.All western
blot experiments were repeated at least three times; in the repeats, separate
cultures were treated, and cell lysates were separately prepared. The band intensities after DOC treatments or
starvation conditions (incubation in HBSS) were then statistically compared
using automated densitometry (QuantiScan Imaging
Analysis, Biology Software Net, UK)
and the values normalized to the control values. Two replicates from the same lysate were also
run to ensure technical reproducibility.


### 2.5. Immunohistochemistry Procedures

Cells were grown in 20 × 100 mm Falcon
polystyrene tissue culture dishes. NCM-460 cells were treated with 0.2 mM DOC for 24 hours. Control (untreated) and DOC-treated cells were
trypsinized, washed with PBS and fixed with 4% formaldehyde overnight. Cell pellets were paraffin-embedded, and 4
micron sections were prepared. The
details of the immunohistochemical procedures have been previously described
[52]. Briefly, after deparaffinization,
rehydration, and incubation in 3% hydrogen peroxide in methanol, sections were
blocked with 1.5% goat serum (Vector Laboratories, Burlingame) and
immunostained using a polyclonal antibeclin-1 antibody from ProSci Inc. (Poway, Calif, USA)
at a concentration of 1 μg/mL. Sections were then incubated using a biotinylated antirabbit secondary
antibody (Vector Laboratories), Vectastain Elite ABC (Avidin Biotin Complex)
reagent (Vector Laboratories), and DAB (3-3′diaminobenzidine) activated by
hydrogen peroxide. Sections were then
counterstained with hematoxylin.


### 2.6. Cytotoxicity Assays

#### 2.6.1. Trypan Blue Exclusion Assay

NCM-460 and apoptosis-resistant HCT-116RC cells were
plated at 2 × 105 cells/mL in a 24 well Falcon plate. After treatments, the supernatants containing
floaters were removed, adherent cells were trypsinized and added to the
floaters. An equal volume of trypan blue
solution was added to a 50 μL
aliquot of the supernatant containing adherent cells and floaters. A minimum of
100 cells were counted on a hemacytometer slide under the 10X objective of a
brightfield microscope. The percentage of cells that were stained with trypan
blue was determined for each treatment. Each experiment was performed at least twice.


#### 2.6.2. Quantitation of Apoptosis and Necrosis

Cells were spun onto Glass slides using the Cytospin
3 (Shandon, Pittsburg,
Pa, USA) and then were fixed in 100%
methanol for 3 minutes. To stain
the slides, 10% Giemsa stain (Sigma) were added for 4 hours. Cells were examined under a 100 X oil
immersion lens and evaluated for apoptosis and necrosis, using criteria
previously reported for brightfield microscopy [21]. All cell death experiments were repeated at
least twice with similar results. To
evaluate statistical significance, 100 cells were scored from 5 different areas
of the slide, and a mean ± S.D. was obtained for each experimental
group. The difference between groups was
considered significant at the 95% probability level using Student’s t-test.


### 2.7. Transmission Electron Microscopy (TEM)

Cells were grown in 10 × 35 mm Falcon
polystyrene tissue culture dishes (Fisher Scientific, Pittsburgh, Pa, USA). All cells were pretreated with protease inhibitors (10μg/mL E-64d, 10 μg/mL pepstatin A) to
enhance the identification of cellular organelles and debris in autophagic
vacuoles. Cells were then incubated with
0.4 mM DOC for 1–3 hours. Control cells
were incubated in the absence of DOC for the same period of time. A total of at least 3 × 106 cells from the two kinds of treatment
groups were rinsed in PBS and fixed in
situ with 1% glutaraldehyde made up in 0.1 M cacodylate buffer (pH 7.2) for
30 minutes. Cells were then scraped from
the surface of the tissue culture plates using a rubber policeman, pelleted, and
then resuspended in 3% glutaraldehyde made up in 0.1 M cacodylate buffer (pH
7.2) for 2 hours at 4°C. Cells were washed in PBS, postfixed in 2% osmium tetroxide, dehydrated
in a graded series of ethanols, and embedded in Spurr’s epoxy resin. Ultrathin sections were stained with uranyl
acetate and lead citrate and photographed using a Philips CM12S transmission
electron microscope operating at 80 keV.


## 2.1. Chemicals

Sodium deoxycholate (DOC), 3-methyladenine
(3-MA), rapamycin (Rapa) (derived from Streptomyces hygroscopicus), CuDIPS
[copper(II) 3,5-diisopropylsalicylate hydrate), and catalase (10 000–40 000 U/mg
protein) were obtained from Sigma-Aldrich (St. Louis, Mo, USA). Bafilomycin A1, MnTBAP [Mn (III)
tetrakis (4-benzoic acid) porphyrin chloride], HBED [N,N′-Di-(2-hydroxybenzyl)ethylenediamine-N,N′-diacetic
acid)], pepstatin A, and E-64d were from BIOMOL Research Laboratories (Plymouth
Meeting, Pa, USA), hydroxychloroquine sulfate (HCS) was from Acros Organics (Morris
Plains, NJ), and trypan blue was from Gibco BRL Life Technologies (Grand Island,
NY, USA). The concentrations used to modulate
autophagy and cell death in the cell culture experiments are provided
below. In all instances, the specific
concentrations that were reported in the literature were tested for their
effect on the viability of NCM-460 and HCT-116RC cells used in the present
study. In some instances dose-response
curves that assess the concentration of the drug and its effect on cell
viability were performed to ensure that the concentration of the chemical itself
was not too toxic to be used in the proposed experiments. This was especially important for 3-MA, which
is used at a wide range of millimolar concentrations in published reports. The three antioxidants (HBED, MnTBAP, CuDIPS)
used to evaluate the DOC-induced increase in beclin-1 expression were used at
the same concentration (100 μM)
so that direct comparisons can be made. The concentration chosen was well within the range of concentrations
reported in the literature for use with cultured cells (see cited articles
below), and this concentration did not induce any cytotoxicity in the sensitive
NCM-460 cells. CuDIPS: 100 μM
[15]; Bafilomycin A1: 1 nM [41, 42]; Catalase: 1000 U/mg;
E-64d: 10 μg/mL [43]; 3-MA: 4 mM; HBED: 100 μM [44, 45]; HCS: 10 μM [46]; MnTBAP: 100 μM [47]; Pepstatin A: 10 μg/mL [43]; Rapa: 100 μM.


## 2.2. Cell Lines and Tissue Culture Conditions

The NCM-460 colon
cell line was obtained from INCELL Corporation, LLC (San Antonio, Tex, USA)
and cultured in M3:10TM medium to insure that it maintains its characteristic
features. This cell line was not
obtained from a colon cancer and is considered to be a noncancerous colonic
epithelial cell line. HCT-116RC is a
stable apoptosis-resistant colon cancer cell line that was developed in our
laboratory after persistent exposure to increasing concentrations of NaDOC
[40]. This cell line was maintained in
DMEM media supplemented with 10% heat-inactivated fetal calf serum (Omega Scientific,
Tarzana, Calif, USA),
1% MEM nonessential amino acids, 100 μg/mL
streptomycin, 100 U/mL penicillin, and 3.44 mg/mL L-glutamine. Media components were from Gibco BRL Life
Technologies (Grand Island,
NY, USA). All treatments utilizing
NCM-460 cells were performed with the cells in approximate mid-logarithmic
phase to avoid inconsistencies in cellular responses based on growth phase, as
previously described [48]. Starvation
experiments were performed by incubation cells in Hank’s Balanced Salt Solution
(HBSS), as previously described for colon epithelial cells [49]. HBSS was obtained from Gibco/Invitrogen Corp., Carlsbad, Calif, USA.


## 2.3. Assessment of Vacuolar Acidification

NCM460 cells were plated on a fibronectin-coated Costar (Fisher Scientific, Pittsburg, Pa, USA)
96 well black plate at 2 × 105 cells/mL. Cells were treated with 0.4 mM DOC for 30 minutes and 1 hour. After
treatments, cells were spun in an Allegra 6R Centrifuge (Beckman Coulter, Fullerton, Calif, USA)
at 1000 rpm for 5 minutes and the fluorescent dyes added as described below.
Triplicate wells were plated for each experimental protocol, and the intensity
values were normalized for each well using a nucleic acid stain. Mean fold
changes in intensity values (DOC versus control untreated cells) were
statistically compared using Student’s t-test.


### 2.3.1. Monodansylcadaverine (MDC)

MDC (Fluka-Sigma-Aldrich, St. Louis, Mo, USA) was used at a final concentration of 200μM, and cells were incubated for 30 minutes at 37°C. MDC was removed and Sytox Green (Molecular
Probes/Invitrogen, Carlsbad,
Calif, USA) was added at a final
concentration of 2.5 μM;
cells were incubated for 20 minutes at room temperature. Fluorescence was assessed immediately using
an Optima FLUOstar plate Reader (BMG Labtech, Durham, NC, USA). MDC (excitation 335 nm; emission 508 nm) and
Sytox Green (excitation 504 nm; emission 523 nm) fluorescence were assessed
using appropriate filters. All
fluorescence values were normalized to the amount of cells in individual wells
by obtaining the ratio of MDC fluorescence (assessment of acid vesicles) to
Sytox Green fluorescence (nucleic acid stain).


### 2.3.2. Lysotracker Red

LysoTracker Red (Molecular Probes/Invitrogen, Carlsbad, Calif, USA) was added to the cells at a final concentration of
100 nM and incubated for 30 minutes at 37°C. Lysotracker Red was removed and Hoechst # 33342 dye (Molecular
Probes/Invitrogen, Carlsbad,
Calif, USA), made up in tissue culture
media at a final concentration of 10μg/mL,
was added to the cells and incubated for 10 minutes at 37°C. Cells were spun at 1000 rpm for 10 minutes,
the supernatant removed, and the pelleted cells were fixed with 4% formaldehyde
in PBS for 20 minutes at room temperature. Fluorescence at 30 minutes and 1 hour was assessed immediately using an
Optima FLUOstar plate Reader (BMG Labtech, Durham, NC, USA). LysoTracker Red (excitation 577 nm; emission
590 nm) and Hoechst (excitation 350 nm; emission 461 nm) fluorescence were
assessed using appropriate filters. All
fluorescence values were normalized to the amount of cells in individual wells
by obtaining the ratio of LysoTracker Red fluorescence (assessment of acid
vesicles) to Hoechst fluorescence (nucleic acid stain).


## 2.3.1. Monodansylcadaverine (MDC)

MDC (Fluka-Sigma-Aldrich, St. Louis, Mo, USA) was used at a final concentration of 200μM, and cells were incubated for 30 minutes at 37°C. MDC was removed and Sytox Green (Molecular
Probes/Invitrogen, Carlsbad,
Calif, USA) was added at a final
concentration of 2.5 μM;
cells were incubated for 20 minutes at room temperature. Fluorescence was assessed immediately using
an Optima FLUOstar plate Reader (BMG Labtech, Durham, NC, USA). MDC (excitation 335 nm; emission 508 nm) and
Sytox Green (excitation 504 nm; emission 523 nm) fluorescence were assessed
using appropriate filters. All
fluorescence values were normalized to the amount of cells in individual wells
by obtaining the ratio of MDC fluorescence (assessment of acid vesicles) to
Sytox Green fluorescence (nucleic acid stain).


## 2.3.2. Lysotracker Red

LysoTracker Red (Molecular Probes/Invitrogen, Carlsbad, Calif, USA) was added to the cells at a final concentration of
100 nM and incubated for 30 minutes at 37°C. Lysotracker Red was removed and Hoechst # 33342 dye (Molecular
Probes/Invitrogen, Carlsbad,
Calif, USA), made up in tissue culture
media at a final concentration of 10μg/mL,
was added to the cells and incubated for 10 minutes at 37°C. Cells were spun at 1000 rpm for 10 minutes,
the supernatant removed, and the pelleted cells were fixed with 4% formaldehyde
in PBS for 20 minutes at room temperature. Fluorescence at 30 minutes and 1 hour was assessed immediately using an
Optima FLUOstar plate Reader (BMG Labtech, Durham, NC, USA). LysoTracker Red (excitation 577 nm; emission
590 nm) and Hoechst (excitation 350 nm; emission 461 nm) fluorescence were
assessed using appropriate filters. All
fluorescence values were normalized to the amount of cells in individual wells
by obtaining the ratio of LysoTracker Red fluorescence (assessment of acid
vesicles) to Hoechst fluorescence (nucleic acid stain).


## 2.4. Western Blot Analysis

Cells
were grown in 20 × 100 mm Falcon polystyrene tissue culture dishes (Fisher
Scientific, Pittsburgh, Pa, USA). Cultures treated with DOC or incubated in control media were disrupted
in lysis buffer (50 mM Tris pH 8, 5 mM EDTA, 150 mM NaCl, 0.5% NP-40) supplemented
with 1 mM phenylmethylsulfonyl fluoride (PMSF), leupeptin (1μg/mL), and aprotinin (0.01 U/mL). Cell lysates were prepared at a concentration
of 2 μg/μL of protein, and 10 μg of protein were added to each well of a 15%
Criterion Tris-HCl gel (Biorad, Hercules, Calif, USA) for
size fractionation by electrophoresis. The proteins were blotted onto
Immobilon-P PVDF transfer membrane (Millipore, Bedford, Mass, USA).
The membranes were incubated with a mouse antibeclin monoclonal antibody (BD
Transduction Laboratories, San Diego, Calif, USA) at a dilution of 1:1000 or rabbit anti-LC3B
polyclonal antibody (Cell Signaling Technology, Inc., Boston, Mass, USA) at a dilution of 1:500. The membranes were then incubated with goat antimouse or goat
antirabbit secondary antibodies conjugated to horseradish peroxidase (Pierce, Rockford, Ill, USA). Antibody complexes were detected using the
SuperSignal West Pico chemiluminescence detection system (Pierce, Rockford, Ill, USA). Finally, the membranes were stained for 20
minutes with Brilliant Blue G dye (Sigma-Aldrich, St. Louis, Mo, USA)
to confirm equal protein loading. We
have chosen to use the staining of the membranes with Brilliant Blue G dye
rather than a specific protein as a loading control, since the former looks at
numerous bands, whereas the latter can be misleading. This is based on work published from our
laboratory using GAPDH and G3PD [50], and those of others who screened 22
housekeeping genes [51] and found a large number to be modulated by various
experimental conditions.All western
blot experiments were repeated at least three times; in the repeats, separate
cultures were treated, and cell lysates were separately prepared. The band intensities after DOC treatments or
starvation conditions (incubation in HBSS) were then statistically compared
using automated densitometry (QuantiScan Imaging
Analysis, Biology Software Net, UK)
and the values normalized to the control values. Two replicates from the same lysate were also
run to ensure technical reproducibility.


## 2.5. Immunohistochemistry Procedures

Cells were grown in 20 × 100 mm Falcon
polystyrene tissue culture dishes. NCM-460 cells were treated with 0.2 mM DOC for 24 hours. Control (untreated) and DOC-treated cells were
trypsinized, washed with PBS and fixed with 4% formaldehyde overnight. Cell pellets were paraffin-embedded, and 4
micron sections were prepared. The
details of the immunohistochemical procedures have been previously described
[52]. Briefly, after deparaffinization,
rehydration, and incubation in 3% hydrogen peroxide in methanol, sections were
blocked with 1.5% goat serum (Vector Laboratories, Burlingame) and
immunostained using a polyclonal antibeclin-1 antibody from ProSci Inc. (Poway, Calif, USA)
at a concentration of 1 μg/mL. Sections were then incubated using a biotinylated antirabbit secondary
antibody (Vector Laboratories), Vectastain Elite ABC (Avidin Biotin Complex)
reagent (Vector Laboratories), and DAB (3-3′diaminobenzidine) activated by
hydrogen peroxide. Sections were then
counterstained with hematoxylin.


## 2.6. Cytotoxicity Assays

### 2.6.1. Trypan Blue Exclusion Assay

NCM-460 and apoptosis-resistant HCT-116RC cells were
plated at 2 × 105 cells/mL in a 24 well Falcon plate. After treatments, the supernatants containing
floaters were removed, adherent cells were trypsinized and added to the
floaters. An equal volume of trypan blue
solution was added to a 50 μL
aliquot of the supernatant containing adherent cells and floaters. A minimum of
100 cells were counted on a hemacytometer slide under the 10X objective of a
brightfield microscope. The percentage of cells that were stained with trypan
blue was determined for each treatment. Each experiment was performed at least twice.


### 2.6.2. Quantitation of Apoptosis and Necrosis

Cells were spun onto Glass slides using the Cytospin
3 (Shandon, Pittsburg,
Pa, USA) and then were fixed in 100%
methanol for 3 minutes. To stain
the slides, 10% Giemsa stain (Sigma) were added for 4 hours. Cells were examined under a 100 X oil
immersion lens and evaluated for apoptosis and necrosis, using criteria
previously reported for brightfield microscopy [21]. All cell death experiments were repeated at
least twice with similar results. To
evaluate statistical significance, 100 cells were scored from 5 different areas
of the slide, and a mean ± S.D. was obtained for each experimental
group. The difference between groups was
considered significant at the 95% probability level using Student’s t-test.


## 2.6.1. Trypan Blue Exclusion Assay

NCM-460 and apoptosis-resistant HCT-116RC cells were
plated at 2 × 105 cells/mL in a 24 well Falcon plate. After treatments, the supernatants containing
floaters were removed, adherent cells were trypsinized and added to the
floaters. An equal volume of trypan blue
solution was added to a 50 μL
aliquot of the supernatant containing adherent cells and floaters. A minimum of
100 cells were counted on a hemacytometer slide under the 10X objective of a
brightfield microscope. The percentage of cells that were stained with trypan
blue was determined for each treatment. Each experiment was performed at least twice.


## 2.6.2. Quantitation of Apoptosis and Necrosis

Cells were spun onto Glass slides using the Cytospin
3 (Shandon, Pittsburg,
Pa, USA) and then were fixed in 100%
methanol for 3 minutes. To stain
the slides, 10% Giemsa stain (Sigma) were added for 4 hours. Cells were examined under a 100 X oil
immersion lens and evaluated for apoptosis and necrosis, using criteria
previously reported for brightfield microscopy [21]. All cell death experiments were repeated at
least twice with similar results. To
evaluate statistical significance, 100 cells were scored from 5 different areas
of the slide, and a mean ± S.D. was obtained for each experimental
group. The difference between groups was
considered significant at the 95% probability level using Student’s t-test.


## 2.7. Transmission Electron Microscopy (TEM)

Cells were grown in 10 × 35 mm Falcon
polystyrene tissue culture dishes (Fisher Scientific, Pittsburgh, Pa, USA). All cells were pretreated with protease inhibitors (10μg/mL E-64d, 10 μg/mL pepstatin A) to
enhance the identification of cellular organelles and debris in autophagic
vacuoles. Cells were then incubated with
0.4 mM DOC for 1–3 hours. Control cells
were incubated in the absence of DOC for the same period of time. A total of at least 3 × 106 cells from the two kinds of treatment
groups were rinsed in PBS and fixed in
situ with 1% glutaraldehyde made up in 0.1 M cacodylate buffer (pH 7.2) for
30 minutes. Cells were then scraped from
the surface of the tissue culture plates using a rubber policeman, pelleted, and
then resuspended in 3% glutaraldehyde made up in 0.1 M cacodylate buffer (pH
7.2) for 2 hours at 4°C. Cells were washed in PBS, postfixed in 2% osmium tetroxide, dehydrated
in a graded series of ethanols, and embedded in Spurr’s epoxy resin. Ultrathin sections were stained with uranyl
acetate and lead citrate and photographed using a Philips CM12S transmission
electron microscope operating at 80 keV.


## 3. Results

### 3.1. DOC Activates the Autophagic Pathway in Noncancer Colonic Epithelial Cells (NCM-460)

Since NCM-460 cells are very sensitive to
DOC-induced cytotoxicity, only 1–3 hour experiments were performed using  0.4 mM
DOC; longer treatment times with this concentration of DOC resulted in
significant apoptosis and necrosis. Treatment of NCM-460 cells with 0.4 mM DOC
for 1–3 hours resulted in the appearance of autophagic vacuoles, assessed using
TEM (Figure1). These ultrastructural findings are considered
one of the gold standards for identifying the activation of autophagy
[53]. Another major finding was the
increase in expression of microtubule-associated proteinlight chain 3 (LC3), the mammalian homologue of the
yeast Atg8 autophagic protein [54]. The
appearance of cytosolic LC3-I by posttranslational modification of Pro-LC3 by
hATG4B [55] and the dynamic increase in the formation of LC3-II (a
LC3-phospholipid conjugate) and localization to autophagosomal membranes [55]
over time in the presence of protease inhibitors (E-64d and pepstatin A) are considered another excellent
indication for the activation of the autophagic pathway [44]. The use of protease inhibitors prevents the
degradation of LC3-II which is membrane-bound and subject to proteolytic
degradation in mature autophagolysosomes. We preincubated NCM-460 cells in media containing 10 μg/mL E-64d and 10 μg/mL pepstatin A for 24 hours and then
exposed cells to 0.4 mM DOC for 0-1 hour, a time period that we determined to
have abundant autophagic vacuoles by TEM and prior to the appearance of
apoptotic or necrotic cells. Figure 2 shows Western blots and
densitometric analysis of DOC-treated NCM-460 cells (Figures 2(a)–2(d)) and starved cells (incubation in HBSS) as a positive
control (Figures 2(e)–2(h)), indicating
the dynamics associated with the appearance of  LC3-I and LC-3-II. It can be seen that both LC3-I and LC3-II
were increased by DOC and starvation conditions at the 1 hour time point
compared to control untreated cells, indicating the activation of the early
cytosolic form of LC3 (LC3-I) and the late membrane-bound form of LC3
(LC3-II). In different experiments the
basal level of LC3-I was more variable than that of LC3-II. The reason for this is not clear, but may
relate to different number of cell passages. In all cases, the basal levels of both LC3-I and LC3-II were increased
by both DOC and starvation conditions; however, the actual fold increase cannot
be directly compared because of this inherent variability.Transmission electron micrographs of control NCM-460 colonic
epithelial cells (a) and cells treated with 0.4 mM DOC for 1 (b), 2 (c), and 3 (d)
hours. Note the increase in number and size of autophagolysosomes after DOC
treatment. (a) Arrow indicates the presence of small, electron-dense lysosomes;
X4,400); (b) arrow indicates a large autophagolysosome with adjacent smaller
autophagolysosomes in the process of fusing with the larger autophagolysosome
(X7,100); (c) a large number of autophagic vacuoles containing cellular debris in
various stages of degradation are present (X7,100); (d) arrow indicates the
presence of a mitochondrion (M) within an autophagic vacuole (X15,000). (All cells were pretreated with protease inhibitors to retard the degradation
process within lysosomes (see Section2); this allowed the
identification of cellular organelles that were difficult to observe in the absence of
the protease inhibitors.) (Uranyl acetate, lead citrate counterstains.)
(a)(b)(c)(d)Western blot analysis of LC3-I and LC3-II protein expressions in NCM-460 cells in the presence of protease inhibitors (see Section2) under
normal conditions, after DOC treatment (a), (c), (d), and under starvation conditions
(e), (g), (h). Bar graphs compare the protein expression of LC3-I and LC3-II over time using computerized densitometric analysis (c), (d), (g), (h).  (a), (c), (d) represent LC3-I and LC3-II protein expressions after incubation with 0.4 mM DOC for 30 and
60 minutes compared to control cells that were not incubated with DOC. (e), (g), (h)
represent LC3-I and LC3-II protein expressions at the same time points under
starvation conditions using HBSS. The membranes were stained with Brilliant
Blue G dye to confirm equal protein loading (Figures 2(b), 2(f)). (∗ indicates
statistically significant (P < .05) differences in mean Relative
Densitometric Units (RDU) between treatment and untreated control groups.)
(a)(b)(c)(d)(e)(f)(g)(h)As shown in Figures2(e)–2(h), the increase of LC3-I and LC3-II over time in the
presence of the protease inhibitors was observed under starvation conditions as
was observed after incubation in 0.4 mM DOC (Figures 2(a)–2(d)). This increase
in LC3-I and LC3-II levels after DOC treatment was similar to the findings of
Ellington et al. [56] who studied soybean B-group triterpenoid saponin-induced
autophagy in a colonic adenocarcinoma cell line (HCT-15). In the present study and that of Ellington et
al. [56], this increase in expression of LC3-I and LC3-II was accompanied by
the presence of autophagic vacuoles assessed by TEM, the classic gold standard
for the activation of the autophagic pathway.An early step in
the autophagic process is the acidification of cytoplasmic vesicles, which
provides the acidic milieu necessary for the optimal activity of digestive
enzymes contained within lysosomes. We
were able to demonstrate the acidification of vesicles within 30 to 60 minutes
after DOC treatment by assessing either the increase in fluorescence of MDC or
Lysotracker Red (Figure3), two dyes that target acid vesicles
[57, 58]. The TEM studies coupled with
the LC3 results and the vesicular acidification assays strongly indicate that
hydrophobic bile acids can activate autophagy as an early stress-response
pathway.Figure 3
Effect of 0.4 mM DOC on the acidification of vesicles using MDC and
Lysotracker Red (LTR) fluorescence assessed with a microplate reader. Note
the statistically significant increase in vesicular acidification within 30 (LTR)–60
(MDC) minutes after DOC treatment. (∗ indicates statistically significant (P < .05)
differences in mean Relative Fluorescence Units (RFU) between treatment
groups and untreated control groups.)DOC also induced
an increase in beclin-1, an essential autophagy protein [59]. NCM-460 cells were exposed to 0.2 mM DOC for
24 hours, and beclin-1 expression was assessed using immunohistochemical (Figure 4(a)) and Western blot (Figures 4(b)–4(d)) analysis. This concentration of DOC did not induce
appreciable apoptosis during a 24-hour period and was, therefore, chosen for
this experiment. Treatment with 0.2 mM DOC
induced a dramatic increase in the protein levels of beclin-1 using both
techniques.Immunohistochemical (a) and Western blot analysis (b), (c) of beclin-1
protein expression in NCM-460 cells in the absence and presence of 0.2 mM
DOC for 24 hours. (a) There is a dramatic increase in the staining of beclin-1 in
both the nucleus and cytoplasm of DOC-treated cells (right panel) compared with
control untreated cells (left panel). Beclin-1 staining also occurs in association
with the nucleolus where a ring of brown staining is observed in several cellular
profiles in DOC-treated cells. (100X oil objective; brown color of DAB indicates
positive beclin-1 staining; blue color indicates hematoxylin stain). (b) Western blot
indicating the increase in the beclin-1 61 kDa band after DOC treatment. (c) The
membranes were stained with Brilliant Blue G dye to confirm equal protein loading for western blot analysis. (d) Computerized densitometric evaluation of
bands shown in (b). (∗ indicates statistically significant (P < .05) differences in
mean Relative Densitometric Units (RDU) between treatment and untreated
control groups.)
(a)(b)(c)(d)


### 3.2. DOC-Induced Increase in Beclin-1 Expression Is Mediated through an Oxidative Stress Mechanism

Since DOC induces a significant amount
of oxidative/nitrosative stress [6, 11–15], we determined if the DOC-induced
increase in beclin-1 expression was mediated, in part, through an oxidative
mechanism. We pretreated NCM-460 cells
for 2 hours with 4 different agents that reduce oxygen-free radicals through
different mechanisms, followed by a 24-hour incubation with 0.2 mM DOC. The  4
agents used were catalase, HBED, MnTBAP, and CuDIPS. Catalase catalytically breaks down hydrogen
peroxide to water and oxygen [60]; HBED is an iron chelator and inhibits ferric
ion catalyzed formation of hydroxyl radicals [45]; MnTBAP is a cell permeable
superoxide dismutase mimetic (SOD) and peroxynitrite scavenger [61, 62]; CuDIPS
is a cell permeable SOD mimetic [63]. All 4 agents had a marked effect on preventing the DOC-induced increase
in beclin-1 expression, although catalase was the most effective (Figure 5). In addition, it was determined that the
constitutive levels of beclin-1 are also highly dependent on endogenous
oxidative stress levels in the cell. As
shown in Figure
5, all 4 antioxidants decreased the constitutive levels of
beclin-1, with catalase being the most effective.Effect of antioxidants and an antioxidant enzyme on protein
expression of beclin-1. Cells were pretreated for 2 hours with either catalase,
HBED, MnTBAP, or CuDIPS, followed by a 24-hour incubation with 0.2 mM DOC.
(a) All pretreatments decreased both the constitutive beclin-1 protein levels and
the DOC-induced increase in beclin-1 protein levels, with catalase being the most
effective. (b) The Brilliant Blue loading control with the position of the beclin-1
band indicated by the arrow.
(a)(b)


### 3.3. Autophagy Protects NCM-460 Cells from DOC-Induced Cell Death

To
determine whether the activation of autophagy contributes to DOC-induced cell
death or is a prosurvival stress-response pathway, NCM-460 cells were
pretreated with rapamycin, an agent that activates autophagy [64], and
3-methyladenine (3-MA), an agent that inhibits the autophagic process [65]. NCM-460 cells were pretreated with 100 μM rapamycin (Figure 6(a)) or 4 mM 3-MA (Figure
6(b)) for 24 hours and then incubated with 0.4 mM DOC for 4 hours. Total cell number and the trypan blue exclusion assay were used as measures
of cell growth and viability.Bar graphs demonstrating the effects of 0.4 mM DOC and 100μM
rapamycin pretreatment (a) or 0.4 mM DOC and 4 mM 3-MA (3-methyladenine)
pretreatment (b) on cell number and viability (trypan blue exclusion) of NCM-460
cells. (Significant differences (P < .05) between treatment groups and control
(untreated) cells are indicated by an asterisk (∗). Significant differences between
DOC treatment, alone, and DOC treatment after rapamycin or 3-MA pretreatment
are indicated by a pound sign (#).)
(a)(b)DOC treatment,
alone, resulted in a significant (P < .05) decrease in cell counts compared
to untreated control cells. Rapamycin
pretreatment significantly (P < .05) decreased trypan blue uptake and
prevented the cell loss caused by DOC treatment (Figure 6(a)). The significant
decrease in cell counts in the absence of significant trypan blue uptake by 100 μM rapamycin, alone (Figure 6(a)), is
most probably a reflection of a decrease in cell proliferation caused by the
activation of autophagy. Opposite to the
effects of rapamycin, pretreatment with 3-MA significantly (P < .05)
increased trypan blue uptake and increased the cell loss caused by DOC (Figure 6(b)).


### 3.4. Autophagy Protects HCT-116RC Apoptosis-Resistant Colon Cancer Cells from DOC-Induced Cell Death

We have previously reported
that persistent exposure of HCT-116 apoptosis-competent colon cancer cells to
increasing concentrations of DOC resulted in the development of stable
apoptosis-resistant cell populations in which several stress-response pathways
were upregulated [40, 66]. It was
determined that the autophagic activity was constitutively upregulated in each
of the apoptosis-resistant cell lines (HCT-116RB, HCT-116RC, HCT-116RD
cells). Increased autophagy was
indicated by the presence of numerous late-stage autophagolysosomes in the
cytoplasm of the resistant cells, identified in some cases by the presence of
numerous whorls of digested material [40].To evaluate   whether the constitutive upregulation of the autophagic pathway has a survival
function in these apoptosis-resistant cells or is merely an epiphenomenon, we
exposed HCT-116RC cells to various agents that modulate the autophagic
process. Since the HCT-116RC cells are
resistant to cell death, all experiments requiring bile acid treatment were
performed using 0.5 mM DOC, and cells were treated in late log phase of
growth. These conditions were necessary
to elicit a cellular response to autophagy inhibitors/inducers, as described
below. HCT-116RC cells were pretreated
with 100μM
rapamycin or 4 mM 3-MA for 24 hours and then incubated with 0.5 mM DOC for an
additional 24 hours. Total cell number
and the trypan blue exclusion assay were used as measures of cell growth and
viability. Similar to the results with
the noncancer cell line, NCM-460, rapamycin pretreatment of the
apoptosis-resistant cancer cell line, HCT-116RC, followed by 24 hours of
treatment with 0.5 mM DOC, resulted in a significant (P < .05) increase in
cell number and a significant (P < .05) decrease in trypan blue uptake
(i.e., increase in viable cells) compared to DOC treatment, alone (Figure 7(a)). The significant decrease in
cell counts in the absence of significant trypan blue uptake by 100 μM
rapamycin, alone (Figure 7(a)), is
most probably a reflection of a decrease in cell proliferation caused by the
activation of autophagy. On the other hand, pretreatment of HCT-116RC
cells with 4 mM 3-MA had no effect on increasing cell death induced by 0.5 mM
DOC (Figure 7(b)). The significant decrease in cell counts in
the absence of significant trypan blue uptake by 4 mM 3-MA, alone, is most
probably a reflection of a decrease in cell proliferation (Figure 7(b)).Bar graphs demonstrating the effects of 0.5 mM DOC and 100μM
rapamycin pretreatment (a) or 0.5 μM DOC and 4 mM 3-MA (3-methyladenine)
pretreatment (b) on cell number and viability (trypan blue exclusion) of HCT 116RC apoptosis-resistant cells. (Significant differences (P < .05) between
treatment groups and control (untreated) cells are indicated by an asterisk (∗).
Significant differences between DOC treatment, alone, and DOC treatment after
rapamycin or 3-MA pretreatment are indicated by a pound sign (#).)
(a)(b)Since we have
previously shown that autophagy is constitutively expressed in these cells [40]
and rapamycin had a significant effect on cell survival, the negative results
obtained with the combination of 3-MA and DOC cannot be taken as conclusive
evidence of lack of involvement of autophagy [58]. Since 3-MA inhibits autophagy at an early
stage by preventing the formation of autophagosomes, it has been reported that
in order to adequately assess the modulation of the autophagy process,
inhibitors that act at a different stages of the autophagy process should also
be tested [58]. Therefore, HCT-116RC
cells were exposed to 2 different inhibitors of the autophagic process,
bafilomycin A1 and hydroxychloroquine, which act at the level of
acid vesicles/lysosomes [67]. Bafilomycin A1 appears to block the
fusion of autophagosomes and lysosomes [68], and hydroxychloroquine (an amine)
diffuses into acid vesicles/lysosomes and raises the intraorganellar pH. Bafilomycin A1 also raises the pH
of acid vesicles/lysosomes by inhibiting the proton-translocating ATPase (H+-ATPase) [69]. HCT-116RC cells were
pretreated with 1 nM bafilomycin A1 for 24 hours and then incubated
with 0.5 mM DOC for an additional 24 hours. Bafilomycin A1 pretreatment followed by DOC treatment
increased the percentage of apoptotic cells 4-fold over the level of apoptosis
induced when DOC was used alone (Table 1). There was no increase in the
percentage of DOC-induced necrotic cells by bafilomycin A1 pretreatment. HCT-116RC cells were
pretreated with 10 μM
hydroxychloroquine for 24 hours and then incubated with 0.5 mM DOC for an
additional 24 hours. Hydroxychloroquine
pretreatment followed by DOC treatment increased the percentage of apoptotic
cells 4-fold over the level of apoptosis induced by DOC, alone (Table 1(b)). There was no increase in the percentage of
DOC-induced necrotic cells by hydroxychloroquine pretreatment.Table 1
(a) Effect of
bafilomycin A1  on DOC-induced
death of apoptosis-resistant
HCT-116RC cells. (b) Effect of hydroxychloroquine on DOC-induced
death of apoptosis-resistant HCT-116RC cells.
(a)
Experimental group% apoptotic cells% necrotic cells(mean ± SEM)(mean ± SEM)Control
(untreated)2.8±1.03.0±0.61 nM Bafilomycin A13.0±0.44.6±0.30.5
mM DOC4.2±0.927.0±3.5*Bafilomycin
A1 + DOC17.8±3.2*#27.8±5.5*(b)
Experimental group% apoptotic cells% necrotic cells(mean ± SEM)(mean ± SEM)Control0.4±0.32.4±0.3*10μM Hydroxychloroquine3.8±1.0*6.4±0.6*0.5 mM DOC5.4±0.9*22.4±4.3*Hydroxychloroquine + DOC20.0±1.7*#19.8±2.6*S*tatistically significant (P < .05) differences in mean values when compared to
untreated control cells; # statistically significant (P < .05) differences in mean
values when compared to cells treated with DOC, alone, and this goes for Tables 1(a) and 1(b).In summary, the
collective data indicate that autophagy has a survival value for both
noncancerous and cancerous colon cells when exposed to hydrophobic bile acids
in a nutrient-rich environment.


## 3.1. DOC Activates the Autophagic Pathway in Noncancer Colonic Epithelial Cells (NCM-460)

Since NCM-460 cells are very sensitive to
DOC-induced cytotoxicity, only 1–3 hour experiments were performed using  0.4 mM
DOC; longer treatment times with this concentration of DOC resulted in
significant apoptosis and necrosis. Treatment of NCM-460 cells with 0.4 mM DOC
for 1–3 hours resulted in the appearance of autophagic vacuoles, assessed using
TEM (Figure1). These ultrastructural findings are considered
one of the gold standards for identifying the activation of autophagy
[53]. Another major finding was the
increase in expression of microtubule-associated proteinlight chain 3 (LC3), the mammalian homologue of the
yeast Atg8 autophagic protein [54]. The
appearance of cytosolic LC3-I by posttranslational modification of Pro-LC3 by
hATG4B [55] and the dynamic increase in the formation of LC3-II (a
LC3-phospholipid conjugate) and localization to autophagosomal membranes [55]
over time in the presence of protease inhibitors (E-64d and pepstatin A) are considered another excellent
indication for the activation of the autophagic pathway [44]. The use of protease inhibitors prevents the
degradation of LC3-II which is membrane-bound and subject to proteolytic
degradation in mature autophagolysosomes. We preincubated NCM-460 cells in media containing 10 μg/mL E-64d and 10 μg/mL pepstatin A for 24 hours and then
exposed cells to 0.4 mM DOC for 0-1 hour, a time period that we determined to
have abundant autophagic vacuoles by TEM and prior to the appearance of
apoptotic or necrotic cells. Figure 2 shows Western blots and
densitometric analysis of DOC-treated NCM-460 cells (Figures 2(a)–2(d)) and starved cells (incubation in HBSS) as a positive
control (Figures 2(e)–2(h)), indicating
the dynamics associated with the appearance of  LC3-I and LC-3-II. It can be seen that both LC3-I and LC3-II
were increased by DOC and starvation conditions at the 1 hour time point
compared to control untreated cells, indicating the activation of the early
cytosolic form of LC3 (LC3-I) and the late membrane-bound form of LC3
(LC3-II). In different experiments the
basal level of LC3-I was more variable than that of LC3-II. The reason for this is not clear, but may
relate to different number of cell passages. In all cases, the basal levels of both LC3-I and LC3-II were increased
by both DOC and starvation conditions; however, the actual fold increase cannot
be directly compared because of this inherent variability.Transmission electron micrographs of control NCM-460 colonic
epithelial cells (a) and cells treated with 0.4 mM DOC for 1 (b), 2 (c), and 3 (d)
hours. Note the increase in number and size of autophagolysosomes after DOC
treatment. (a) Arrow indicates the presence of small, electron-dense lysosomes;
X4,400); (b) arrow indicates a large autophagolysosome with adjacent smaller
autophagolysosomes in the process of fusing with the larger autophagolysosome
(X7,100); (c) a large number of autophagic vacuoles containing cellular debris in
various stages of degradation are present (X7,100); (d) arrow indicates the
presence of a mitochondrion (M) within an autophagic vacuole (X15,000). (All cells were pretreated with protease inhibitors to retard the degradation
process within lysosomes (see Section2); this allowed the
identification of cellular organelles that were difficult to observe in the absence of
the protease inhibitors.) (Uranyl acetate, lead citrate counterstains.)
(a)(b)(c)(d)Western blot analysis of LC3-I and LC3-II protein expressions in NCM-460 cells in the presence of protease inhibitors (see Section2) under
normal conditions, after DOC treatment (a), (c), (d), and under starvation conditions
(e), (g), (h). Bar graphs compare the protein expression of LC3-I and LC3-II over time using computerized densitometric analysis (c), (d), (g), (h).  (a), (c), (d) represent LC3-I and LC3-II protein expressions after incubation with 0.4 mM DOC for 30 and
60 minutes compared to control cells that were not incubated with DOC. (e), (g), (h)
represent LC3-I and LC3-II protein expressions at the same time points under
starvation conditions using HBSS. The membranes were stained with Brilliant
Blue G dye to confirm equal protein loading (Figures 2(b), 2(f)). (∗ indicates
statistically significant (P < .05) differences in mean Relative
Densitometric Units (RDU) between treatment and untreated control groups.)
(a)(b)(c)(d)(e)(f)(g)(h)As shown in Figures2(e)–2(h), the increase of LC3-I and LC3-II over time in the
presence of the protease inhibitors was observed under starvation conditions as
was observed after incubation in 0.4 mM DOC (Figures 2(a)–2(d)). This increase
in LC3-I and LC3-II levels after DOC treatment was similar to the findings of
Ellington et al. [56] who studied soybean B-group triterpenoid saponin-induced
autophagy in a colonic adenocarcinoma cell line (HCT-15). In the present study and that of Ellington et
al. [56], this increase in expression of LC3-I and LC3-II was accompanied by
the presence of autophagic vacuoles assessed by TEM, the classic gold standard
for the activation of the autophagic pathway.An early step in
the autophagic process is the acidification of cytoplasmic vesicles, which
provides the acidic milieu necessary for the optimal activity of digestive
enzymes contained within lysosomes. We
were able to demonstrate the acidification of vesicles within 30 to 60 minutes
after DOC treatment by assessing either the increase in fluorescence of MDC or
Lysotracker Red (Figure3), two dyes that target acid vesicles
[57, 58]. The TEM studies coupled with
the LC3 results and the vesicular acidification assays strongly indicate that
hydrophobic bile acids can activate autophagy as an early stress-response
pathway.Figure 3
Effect of 0.4 mM DOC on the acidification of vesicles using MDC and
Lysotracker Red (LTR) fluorescence assessed with a microplate reader. Note
the statistically significant increase in vesicular acidification within 30 (LTR)–60
(MDC) minutes after DOC treatment. (∗ indicates statistically significant (P < .05)
differences in mean Relative Fluorescence Units (RFU) between treatment
groups and untreated control groups.)DOC also induced
an increase in beclin-1, an essential autophagy protein [59]. NCM-460 cells were exposed to 0.2 mM DOC for
24 hours, and beclin-1 expression was assessed using immunohistochemical (Figure 4(a)) and Western blot (Figures 4(b)–4(d)) analysis. This concentration of DOC did not induce
appreciable apoptosis during a 24-hour period and was, therefore, chosen for
this experiment. Treatment with 0.2 mM DOC
induced a dramatic increase in the protein levels of beclin-1 using both
techniques.Immunohistochemical (a) and Western blot analysis (b), (c) of beclin-1
protein expression in NCM-460 cells in the absence and presence of 0.2 mM
DOC for 24 hours. (a) There is a dramatic increase in the staining of beclin-1 in
both the nucleus and cytoplasm of DOC-treated cells (right panel) compared with
control untreated cells (left panel). Beclin-1 staining also occurs in association
with the nucleolus where a ring of brown staining is observed in several cellular
profiles in DOC-treated cells. (100X oil objective; brown color of DAB indicates
positive beclin-1 staining; blue color indicates hematoxylin stain). (b) Western blot
indicating the increase in the beclin-1 61 kDa band after DOC treatment. (c) The
membranes were stained with Brilliant Blue G dye to confirm equal protein loading for western blot analysis. (d) Computerized densitometric evaluation of
bands shown in (b). (∗ indicates statistically significant (P < .05) differences in
mean Relative Densitometric Units (RDU) between treatment and untreated
control groups.)
(a)(b)(c)(d)


## 3.2. DOC-Induced Increase in Beclin-1 Expression Is Mediated through an Oxidative Stress Mechanism

Since DOC induces a significant amount
of oxidative/nitrosative stress [6, 11–15], we determined if the DOC-induced
increase in beclin-1 expression was mediated, in part, through an oxidative
mechanism. We pretreated NCM-460 cells
for 2 hours with 4 different agents that reduce oxygen-free radicals through
different mechanisms, followed by a 24-hour incubation with 0.2 mM DOC. The  4
agents used were catalase, HBED, MnTBAP, and CuDIPS. Catalase catalytically breaks down hydrogen
peroxide to water and oxygen [60]; HBED is an iron chelator and inhibits ferric
ion catalyzed formation of hydroxyl radicals [45]; MnTBAP is a cell permeable
superoxide dismutase mimetic (SOD) and peroxynitrite scavenger [61, 62]; CuDIPS
is a cell permeable SOD mimetic [63]. All 4 agents had a marked effect on preventing the DOC-induced increase
in beclin-1 expression, although catalase was the most effective (Figure 5). In addition, it was determined that the
constitutive levels of beclin-1 are also highly dependent on endogenous
oxidative stress levels in the cell. As
shown in Figure
5, all 4 antioxidants decreased the constitutive levels of
beclin-1, with catalase being the most effective.Effect of antioxidants and an antioxidant enzyme on protein
expression of beclin-1. Cells were pretreated for 2 hours with either catalase,
HBED, MnTBAP, or CuDIPS, followed by a 24-hour incubation with 0.2 mM DOC.
(a) All pretreatments decreased both the constitutive beclin-1 protein levels and
the DOC-induced increase in beclin-1 protein levels, with catalase being the most
effective. (b) The Brilliant Blue loading control with the position of the beclin-1
band indicated by the arrow.
(a)(b)


## 3.3. Autophagy Protects NCM-460 Cells from DOC-Induced Cell Death

To
determine whether the activation of autophagy contributes to DOC-induced cell
death or is a prosurvival stress-response pathway, NCM-460 cells were
pretreated with rapamycin, an agent that activates autophagy [64], and
3-methyladenine (3-MA), an agent that inhibits the autophagic process [65]. NCM-460 cells were pretreated with 100 μM rapamycin (Figure 6(a)) or 4 mM 3-MA (Figure
6(b)) for 24 hours and then incubated with 0.4 mM DOC for 4 hours. Total cell number and the trypan blue exclusion assay were used as measures
of cell growth and viability.Bar graphs demonstrating the effects of 0.4 mM DOC and 100μM
rapamycin pretreatment (a) or 0.4 mM DOC and 4 mM 3-MA (3-methyladenine)
pretreatment (b) on cell number and viability (trypan blue exclusion) of NCM-460
cells. (Significant differences (P < .05) between treatment groups and control
(untreated) cells are indicated by an asterisk (∗). Significant differences between
DOC treatment, alone, and DOC treatment after rapamycin or 3-MA pretreatment
are indicated by a pound sign (#).)
(a)(b)DOC treatment,
alone, resulted in a significant (P < .05) decrease in cell counts compared
to untreated control cells. Rapamycin
pretreatment significantly (P < .05) decreased trypan blue uptake and
prevented the cell loss caused by DOC treatment (Figure 6(a)). The significant
decrease in cell counts in the absence of significant trypan blue uptake by 100 μM rapamycin, alone (Figure 6(a)), is
most probably a reflection of a decrease in cell proliferation caused by the
activation of autophagy. Opposite to the
effects of rapamycin, pretreatment with 3-MA significantly (P < .05)
increased trypan blue uptake and increased the cell loss caused by DOC (Figure 6(b)).


## 3.4. Autophagy Protects HCT-116RC Apoptosis-Resistant Colon Cancer Cells from DOC-Induced Cell Death

We have previously reported
that persistent exposure of HCT-116 apoptosis-competent colon cancer cells to
increasing concentrations of DOC resulted in the development of stable
apoptosis-resistant cell populations in which several stress-response pathways
were upregulated [40, 66]. It was
determined that the autophagic activity was constitutively upregulated in each
of the apoptosis-resistant cell lines (HCT-116RB, HCT-116RC, HCT-116RD
cells). Increased autophagy was
indicated by the presence of numerous late-stage autophagolysosomes in the
cytoplasm of the resistant cells, identified in some cases by the presence of
numerous whorls of digested material [40].To evaluate   whether the constitutive upregulation of the autophagic pathway has a survival
function in these apoptosis-resistant cells or is merely an epiphenomenon, we
exposed HCT-116RC cells to various agents that modulate the autophagic
process. Since the HCT-116RC cells are
resistant to cell death, all experiments requiring bile acid treatment were
performed using 0.5 mM DOC, and cells were treated in late log phase of
growth. These conditions were necessary
to elicit a cellular response to autophagy inhibitors/inducers, as described
below. HCT-116RC cells were pretreated
with 100μM
rapamycin or 4 mM 3-MA for 24 hours and then incubated with 0.5 mM DOC for an
additional 24 hours. Total cell number
and the trypan blue exclusion assay were used as measures of cell growth and
viability. Similar to the results with
the noncancer cell line, NCM-460, rapamycin pretreatment of the
apoptosis-resistant cancer cell line, HCT-116RC, followed by 24 hours of
treatment with 0.5 mM DOC, resulted in a significant (P < .05) increase in
cell number and a significant (P < .05) decrease in trypan blue uptake
(i.e., increase in viable cells) compared to DOC treatment, alone (Figure 7(a)). The significant decrease in
cell counts in the absence of significant trypan blue uptake by 100 μM
rapamycin, alone (Figure 7(a)), is
most probably a reflection of a decrease in cell proliferation caused by the
activation of autophagy. On the other hand, pretreatment of HCT-116RC
cells with 4 mM 3-MA had no effect on increasing cell death induced by 0.5 mM
DOC (Figure 7(b)). The significant decrease in cell counts in
the absence of significant trypan blue uptake by 4 mM 3-MA, alone, is most
probably a reflection of a decrease in cell proliferation (Figure 7(b)).Bar graphs demonstrating the effects of 0.5 mM DOC and 100μM
rapamycin pretreatment (a) or 0.5 μM DOC and 4 mM 3-MA (3-methyladenine)
pretreatment (b) on cell number and viability (trypan blue exclusion) of HCT 116RC apoptosis-resistant cells. (Significant differences (P < .05) between
treatment groups and control (untreated) cells are indicated by an asterisk (∗).
Significant differences between DOC treatment, alone, and DOC treatment after
rapamycin or 3-MA pretreatment are indicated by a pound sign (#).)
(a)(b)Since we have
previously shown that autophagy is constitutively expressed in these cells [40]
and rapamycin had a significant effect on cell survival, the negative results
obtained with the combination of 3-MA and DOC cannot be taken as conclusive
evidence of lack of involvement of autophagy [58]. Since 3-MA inhibits autophagy at an early
stage by preventing the formation of autophagosomes, it has been reported that
in order to adequately assess the modulation of the autophagy process,
inhibitors that act at a different stages of the autophagy process should also
be tested [58]. Therefore, HCT-116RC
cells were exposed to 2 different inhibitors of the autophagic process,
bafilomycin A1 and hydroxychloroquine, which act at the level of
acid vesicles/lysosomes [67]. Bafilomycin A1 appears to block the
fusion of autophagosomes and lysosomes [68], and hydroxychloroquine (an amine)
diffuses into acid vesicles/lysosomes and raises the intraorganellar pH. Bafilomycin A1 also raises the pH
of acid vesicles/lysosomes by inhibiting the proton-translocating ATPase (H+-ATPase) [69]. HCT-116RC cells were
pretreated with 1 nM bafilomycin A1 for 24 hours and then incubated
with 0.5 mM DOC for an additional 24 hours. Bafilomycin A1 pretreatment followed by DOC treatment
increased the percentage of apoptotic cells 4-fold over the level of apoptosis
induced when DOC was used alone (Table 1). There was no increase in the
percentage of DOC-induced necrotic cells by bafilomycin A1 pretreatment. HCT-116RC cells were
pretreated with 10 μM
hydroxychloroquine for 24 hours and then incubated with 0.5 mM DOC for an
additional 24 hours. Hydroxychloroquine
pretreatment followed by DOC treatment increased the percentage of apoptotic
cells 4-fold over the level of apoptosis induced by DOC, alone (Table 1(b)). There was no increase in the percentage of
DOC-induced necrotic cells by hydroxychloroquine pretreatment.Table 1
(a) Effect of
bafilomycin A1  on DOC-induced
death of apoptosis-resistant
HCT-116RC cells. (b) Effect of hydroxychloroquine on DOC-induced
death of apoptosis-resistant HCT-116RC cells.
(a)
Experimental group% apoptotic cells% necrotic cells(mean ± SEM)(mean ± SEM)Control
(untreated)2.8±1.03.0±0.61 nM Bafilomycin A13.0±0.44.6±0.30.5
mM DOC4.2±0.927.0±3.5*Bafilomycin
A1 + DOC17.8±3.2*#27.8±5.5*(b)
Experimental group% apoptotic cells% necrotic cells(mean ± SEM)(mean ± SEM)Control0.4±0.32.4±0.3*10μM Hydroxychloroquine3.8±1.0*6.4±0.6*0.5 mM DOC5.4±0.9*22.4±4.3*Hydroxychloroquine + DOC20.0±1.7*#19.8±2.6*S*tatistically significant (P < .05) differences in mean values when compared to
untreated control cells; # statistically significant (P < .05) differences in mean
values when compared to cells treated with DOC, alone, and this goes for Tables 1(a) and 1(b).In summary, the
collective data indicate that autophagy has a survival value for both
noncancerous and cancerous colon cells when exposed to hydrophobic bile acids
in a nutrient-rich environment.


## 4. Discussion

High concentrations of hydrophobic bile
acids, associated with a high-fat diet, induce proapoptotic and prosurvival
stress-response pathways [13]. The
ultimate fate of the cell depends upon the balance of proapoptotic and
antiapoptotic proteins activated or synthesized in response to bile acid exposure,
and the level of energy demands placed upon the stressed cell [9]. We hypothesized that persistent cellular
stress induced by bile acids, such as ER stress, DNA damage, and mitochondrial
stress, will lead to the clonal selection of apoptosis-resistant cells and the
constitutive activation of cell survival pathways (Figure 8). We tested this
hypothesis by generating apoptosis-resistant colon cells by repeated  exposure
of apoptosis-sensitive HCT-116 cells in
vitro to increasing concentrations of the hydrophobic bile acid, DOC, and
evaluating stress-induced cell death and apoptosis-related gene expression at
the molecular and cellular levels [40, 66]. NF-κB
and many proteins that protect against oxidative stress were constitutively
upregulated in these apoptosis-resistant cells. In addition, the development of apoptosis
resistance was accompanied by the modulation of genes associated with the
autophagy pathway. The autophagy-related genes that exhibit increased
expression include six rab genes involved in vesicle transport, a Rab
interacting lysosomal protein-like 2 protein (RILPL2), PI(3)K, 2 subunits of the
lysosomal proton (H+)-translocating ATPase, cathepsin D, lysosomal-associated membrane protein 1
(Lamp-1), a multipass membrane transporter protein (MFSD8/CLN7), and
prenylcysteine lyase, a lysosomal enzyme involved in the degradation of
prenylated proteins. We also found that
chronic feeding of wild-type B6.129 mice with DOC added to the diet results in
an increase in APG4 [17], a cysteine protease that acts during the formation of
autophagosomes [70] and whose activity is regulated by reactive oxygen species
(ROS) [71]. The functional role of
autophagy in colon carcinogenesis, however, was not determined from these in vitro microarray and in vivo animal studies.Figure 8
Diagram indicating the possible roles of autophagy in colon
carcinogenesis. Hydrophobic bile acids are known to induce numerous stresses
in colon epithelial cells that result in the activation of prosurvival stress-response
pathways. NF-κB activation by hydrophobic bile acids induces prosurvival
pathways. The present study reports that deoxycholate (DOC), a hydrophobic
bile acid that is important in colon carcinogenesis, activates autophagy. This
activation of autophagy by DOC was also shown to have a prosurvival function.
The constitutive upregulation of prosurvival pathways (resulting from chronic
exposure to DOC and selection for apoptosis resistance) can enhance mutation
rates, which may lead to the development of colon cancer. NCM-460 and HCT-
116RC cells were used as model cell lines to evaluate the bile acid-induced
activation of the autophagic pathway and its consequences in the early and late
stages of colon carcinogenesis, respectively.In the
present study, we first evaluated the ability of DOC to activate autophagy in
NCM-460 cells, and then determined whether autophagy has a prosurvival
function in this noncancerous colon epithelial cell line. We demonstrated that DOC activated autophagy
using different methods of detection, and that this activation contributed to
cell survival. We next determined that
the constitutive upregulation of autophagy also has a prosurvival function in
HCT-116RC apoptosis-resistant colon cancer epithelial cells. The prosurvival mechanism
of DOC-induced autophagy is probably antiapoptotic. This is based on the
experiments with bafilomycin A1/hydroxychloroquine, in which we
showed that autophagy prevented cells from undergoing DOC-induced apoptosis,
but not from  DOC-induced necrosis.The
possible roles of autophagy in colon carcinogenesis based on published results
and present findings are shown schematically in Figure8. The cellular stresses induced by hydrophobic
bile acids (e.g., ER stress, DNA damage, mitochondrial stress) are also inducers
of the autophagic pathway [72–75], most probably mediated through the
generation of ROS [76]. Evidence that
DOC induces the autophagic pathway through an oxidative/nitrosative mechanism
was provided in the present study using 3 different antioxidants in addition to
catalase. These antioxidant conditions
dramatically reduced the level of DOC-induced beclin-1 protein expression, a
major protein involved in the mammalian autophagic pathway.The
rationale for choosing beclin-1 (homologue of the yeast autophagy gene
apg6/vps30 [77]) to assess the effects of
antioxidants on the DOC-induced increase in autophagy was based on (1)
its dramatic increase in expression in noncancer cells by DOC, a known inducer
of oxidative stress, compared to other autophagy-related proteins (data not
shown), (2) its critical involvement in the initial step of autophagosome
formation [78–80], (3) the documented importance of an increase in beclin-1 at
the premetastasis stage of colon cancer development [81], (4), its role in
tumorigenesis, in general [82, 83], (5) its function as an antiapoptotic protein
[84–87], and (6) its potential as a possible biomarker to assess colon cancer
risk. Although the oxidative mechanism
by which DOC increases beclin-1 protein expression is most probably
multifactorial, we suggest that an important signaling pathway may involve the
generation of ceramide. This is based on
the fact that (1) ceramide is an important sphingolipid molecule involved in the
increase in beclin-1 in HT-29 colon epithelial cells [88] and other cell types
[89], DOC is known to generate ceramide through several mechanisms [80–92], (2) ceramide
treatment decreases catalase enzymatic activity and expression [93], a possible
link to the present findings indicating that catalase reduces the DOC-induced
increase in beclin-1 expression, and (3) ceramide can damage mitochondria
[94–98], a known inducer of the autophagic process [99]. Other mechanisms that may be responsible for
DOC-induced increase in beclin-1 expression may involve alterations of lipid
trafficking [100], a process known to induce beclin-1 expression in other cell
types [101]. Although we focussed on the
role of oxidative stress in the DOC-induced modulation of beclin-1, other
aspects of the autophagic process that are now known to be regulated by ROS/RNS [102–105] may also be
modulated by DOC.Since we have shown that autophagy is a
survival pathway for apoptosis-resistant colon cancer cells, the constitutive
activation of autophagy and the activation of autophagy induced by cancer
chemotherapeutic agents [106] should, therefore, be taken into consideration
when designing effective clinical treatment regimens for cancer. We plan to determine the effectiveness of
modulators of autophagy in combination with cytotoxic drugs, such as
5-fluorouracil, oxaliplatin, and irenotecan [107, 108], in enhancing cell death in vitro in our apoptosis-resistant
colon cancer cell lines. The precedence
for combining inhibitors of autophagy with chemotherapeutic agents for the
treatment of colon was recently established [106]. Li et al. [106] reported that 3-MA enhanced
the effect of 5-fluorouracil in inducing apoptosis of colo26 and HT-29 colon
cancer cells. It is also anticipated
that a better understanding of the mechanisms of autophagy in colon cells,
their modulation by dietary factors, and aberrant expression of autophagic
proteins during colon carcinogenesis will contribute to the important field of
hypothesis-driven biomarker development to assess colon cancer risk.



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