Prostate cancer cell lines and tumor tissues have been shown to possess upregulated quantities of reactive oxygen species (ROS) (Lim et al., 2005) and these ROS are becoming increasingly associated with several aspects of prostate cancer progression including not only carcinogenesis but also tumor cell proliferation and invasion. In prostate cancer, oxygen radicals are reported to arise from several sources within the cells including the NADPH oxidase (Lim et al., 2005), mitochondrial glycerophosphate-dependent ROS (Chowdhury et al., 2005), xanthine oxidase and nitric oxide synthases (Chiarugi, 2003). The cells net redox state is a balance between oxygen radical synthesis and breakdown, and net ROS generation in prostate cancer has also been reported to develop from downregulated levels or activities of the scavenger enzyme systems catalase, superoxide dismutase I (Zn 2+/Cu 2+ SOD) and II (MN-SOD), and glutathione peroxidase (Chiarugi, 2003).
While most attention has focused on ROS mediation of carcinogenesis and proliferation,
some evidence also associates ROS with tumor invasion and metastasis. In metastatic
cell lines derived from the human prostate cancer line LNCaP, Lim (2005) reports
elevated levels of nox1 and H2O2. Some potential targets
of ROS activity during invasion have been identified. For instance, in the human
prostate cancer line PC-3, ROS appear to regulate matrix metalloproteinase-2
(MMP-2), a type IV collagenase which degrades the basement membrane (Shariftabrizi
et al., 2005). Oxygen radicals in this cell line also induce VEGF which
can promote angiogenesis (Gao et al., 2004). ROS may also serve as second
messengers to mediate adhesive signals from integrins or cadherins to the focal
adhesion kinase (FAK) (Chiargi, 2003). Although adhesive signaling may contribute
to enhanced migration, a critical role of ROS in prostate cancer cell motility
and migration has not been previously reported.
In this study, we wished to first determine if ROS varied as a function of prostate cancer cell density. We found that low cell density is associated with the generation of ROS. The behavior of ROS-positive cells in culture suggested that ROS may influence the migration of cells away from tumor cell clusters and therefore we assessed the effect of ROS on migration, adhesion to specific matrix substrates and in vitro invasion. We also compared the activities of two major ROS catabolic pathways and the glutathione redox ratio between low and high density cells. Present results link increased ROS in relatively isolated prostate cancer cells with in vitro migration and invasion.
Materials and Methods
Cell Culture and Treatments
PC3M cells were a gracious gift from Dr. Isiah Fidler (MD Anderson Cancer
Center, Houston TX). These cells were maintained in Minimal Essential Medium
with Earles salts (MEM), supplemented with 10% fetal bovine serum, 100
units mL-1 penicillin, 100 ug mL-1 streptomycin, and 2.0
mM L-glutamine. To detect reactive oxygen species, cells were treated with 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein
diacetate, acetyl ester (CM-H2DCFDA, purchased from Molecular Probes).
This probe is a cell permeant ester which is hydrolyzed in the cytoplasm of
live cells. When oxidized by molecules such as superoxide anion or hydroxyl
radical, CM-H2DCFDA fluoresces with excitation/emission spectra peaks
at 495 and 525 nm, respectively. To load CM-H2DCFDA into cells, monolayer
cultures were washed using Hanks Balanced Salt solution with calcium and
magnesium (HBSS-Ca, Mg), then incubated at 37°°C for one
hour in a solution of HBSS-Ca, Mg containing 10 uM CM-H2DCFDA. Cells
were then washed in two consecutive changes of MEM containing 0.1% bovine serum
albumin (BSA) for 30 min each. Following washes, cells were viewed and imaged
under epifluorescence with a Zeiss Axioplan 200 microscope using an FITC-Alexafluor
488 compatible filter set (Chroma Filters No. 41017); images were captured with
an Olympus Q Capture 5 camera and Q Capture Pro software. Quantitative fluorescence
measurements were obtained using a Spectra Max 2 fluorescent spectrophotometer
at excitation 485 and emission 530 nm. Fluorescence intensity (in units) was
divided by the cell number in each well to yield an intensity per 10,000 cells.
For ROS inhibition studies, two compounds were used: the SOD mimetic 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy, free radical (TEMPOL, purchased from Sigma Chemicals) and N-acetyl cysteine (NAC, Sigma Chemicals), a more water soluble form of the amino acid cysteine which is a component of the antioxidant glutathione. Tempol was diluted in HBSS-Ca, Mg and used at a final concentration of 2.0 mM; NAC was diluted in HBSS, neutralized to pH 7.0 and used at a final concentration of 2.0 mM.
To quantitate the horizontal movement of cells, the scratch assay was used.
For this assay, a confluent monolayer of cells is established and then a scratch
is made through the monolayer in an X pattern using a standard plastic
1-200 uL pipet tip. This creates an in vitro wound approximately 700
um wide. Cancer cells fill in the scratch area by migrating as single cells
from the confluent sides. The width of the scratch was measured using Q-Capture
Pro software each day until the gap filled in completely. Eight replicate wells
from a 24-well plate were used for each experimental condition. The scratch
gap in each well was measured in four separate areas and these measurements
were averaged to give the overall measurement for that well. Data was analyed
using linear regression (GraphPad Prism software, San Diego, CA).
To quantitate vertical motility, an in vitro motility assay was used. In this assay, FluoroBlok transwell inserts with 8 uM pores (BD Falcon) were inserted into the top of 24-well tissue culture plates to create a two-chamber culture. PC3M cells (105 total) were plated into the upper chamber and allowed to migrate vertically through the pores to the lower chamber. In this motility assay, both upper and lower chambers will filled with maintenance culture media and after one day, cells which migrated through the pores to the lower membrane surface were visualized and counted. To easily identify the cells on the lower membrane surface, the transwell insert was incubated in a 100 uM solution of Calcein Blue fluorescent indicator (Molecular Probes) diluted in HBSS. After one hour, the calcein Blue removed using two fifteen minutes. incubations of the insert with HBSS. This treatment produced a bright blue fluorescence in live cells on the lower membrane while the FluoroBlok coating blocked fluorescence coming from the cells in the upper chamber.
To assay in vitro invasion, Fluoroblok inserts were pre-coated with a 100 uL solution of Matrigel diluted in growth medium to 1 mg mL-1. Matrigel was allowed to dry onto the upper surface of the membrane overnight to evenly coat the surface. One-hundred thousand PC3M cells were plated into the upper chamber as described above and quantitated after two days using Calcein Blue dye uptake. Data for motility and invasion assays were analyzed by t-test.
Cells were plated at an initial concentration of 10,000 cells per well in
24-well plates in maintenance media and treated with tempol or NAC at 2 mM each.
At two day intervals, cells from four replicate wells for control and each treatment
were counted using a hemocytometer.
Twenty-four well tissue culture plates were coated overnight with 20 ug
well of human fibronectin or vitronectin (both purchased from Sigma Chemicals),
Matrigel (BD Biosciences, San Jose, CA) or type IV collagen (Rockland Immunochemicals,
Gilbertsville, PA). The coated wells were washed twice with PBS then blocked
with 1 mL of 1% BSA in PBS for 1 h at 37°C. Following a second PBS wash,
1x105 prostate cancer cells were seeded onto the matrices or into
BSA only-blocked wells for a background measurement for 1 h at 37°C. Non-adherant
cells were removed by aspiration and all wells were then washed twice with PBS.
Adherent cells were removed by trypsinization and counted. Results are the mean
and standard deviation of four wells for each cell line and inhibitor-treated
populations were compared to untreated populations using one-way ANOVA.
Glutathione Ratio (Reduced Glutathione/Oxidized Glutathione)
Detection and quantification of total and oxidized glutathione was performed
as described (Anderson, 1985, Griffith, 1980). Briefly, to assay total glutathione,
a 50 μL aliquot of protein sample (cell lysate) was combined with 700 μL
0.298 mM NADPH, 100 μL Ellmans reagent, 100 μL dH2O, and 50
μL glutathione reductase. Standards were prepared in the same manner, substituting
50 μL 5% 5-sulfosalicylic acid in place of sample. Chromogenic product
formation was quantitated at 412 nm using a Multiskan Spectrum spectrophotometer
(Thermo Electron Corporation, Waltham, MA). All chemicals were purchased from
Sigma-Aldrich, St. Louis, MO. To assay oxidized glutathione, a 30 μL aliquot
of cell lysate was mixed with 2 uL 2-viynlpyridine and incubated at room temperature
for sixty minutes. A 5% solution of 5-sulfosalicylic acid (30 μL) was then
added to the lysate and samples were analyzed as for total glutathione. Reduced
glutathione content was calculated by subtracting oxidized glutathione from
total glutathione. Final values were calculated based on equivalent amounts
of total protein.
Superoxide Dismutase (SOD) Assay
SOD activity was measured using an SOD Assay Kit according to the manufacturers
protocol (Dojindo Molecular Technologies, Inc., Gaithersburg, MD).
To determine catalase activity, cells were suspended in 50mM potassium phosphate
buffer pH 7.0 and sonicated. Aliquots of cell homogenate (2-5 mg total protein
mL-1) were activated with 30 mM H2O2, and then
assayed at 240 nm every 30 sec for 2 min. This assay has been previously described
(Aebi, 1984; Beers and Sizer, 1952).
ROS Levels Become Increased in Prostate Cancer Cells at Lower Densities
PC3M cancer cells were plated at 5000 cells/well or 150,000 cells/well in
24-well plates and allowed to attach overnight. Accumulated ROS were visualized
by loading cells with CM-H2DCFDA indicator, which generated green
fluorescence upon oxidation. At the low density level, approximately 80% of
cells were visibly green (Fig. 1A, phase contrast image shows
all cells present in field of view). Cells at high density, contained less than
15% of cells with ROS fluorescence (Fig. 1B). The intensity
of green fluorescence varied somewhat in the population; however ROS positive
cells at low density setting appeared to be flat and spreading while intense
ROS positive cells in the confluent setting were rounded.
To quantify the inverse association of ROS with density, PC3M cells were plated at various densities using serial dilution beginning with 150,000 cells/well. Fluorescence was quantified and is expressed on a cell basis (Fig. 1C). This experiment demonstrated a marked increase in cellular ROS as the density decreased. The level of fluorescence appeared to plateau at densities below 10,000 cells/well (approximately 5000 cells cm-2).
PC3M Migration In Vitro Can Be Modulated by Inhibiting and Enhancing Reactive
The appearance of increased ROS in cells at low density suggests that the
reactive oxygen might play a role in motility, adhesion or proliferation (Mooney
et al., 1992). To determine whether ROS production is involved in migration,
we investigated the effect of reducing net ROS utilizing tempol and NAC in a
In this assay, PC3M cells were plated to confluence, treated with inhibitors,
and then the monolayer surface of cells was scratched. The cancer cells migrate
across the gap from both sides filling in the area (Fig. 2A,
upper panel). Individual cells which left the confluent population lining the
gap and migrated into the space showed a notable incidence of ROS-induced fluorescence
compared with cells on the side (Fig. 2A, middle and lower
panel). To measure the effect of inhibiting ROS on this horizontal migration,
cells were treated with tempol and NAC and the width of the gap was measured
on a daily basis.
||Reactive Oxygen Species (ROS) in PC3M cells at low density
|Green fluorescence indicative of ROS oxidation of intracellular
CM-H2DCFDA indicator in cells plated at 5000 cells/well (100
x magnification). Fluorescent cells are shown (left frame) compared to a
phase contrast image of all cells in the field of view (right frame)
|Green ROS fluorescence in high density cells. Fluorescent cells only (left
frame), phase contrast of all cells (right frame)
|Fluorescent intensity from ROS increases as cell density decreases. PC3M
cells were plated in 24-well plates at four dilutions. The cells were then
loaded with CM-H2DCFDA indicator and assayed using fluorescence
spectroscopy. Results are expressed as fluorescence units per 10,000 cells
vs. final cell density. Values represent mean of 4 replicates ± SEM
||Decreased rates of migration are associated with inhibition
of ROS by tempol and NAC
| Upper panel: Phase contrast images (100x) show the time course
of cellular migration resulting in filling of the gap created in the scratch
assay. Middle panel: Cells which have migrated into the gap show a greater
incidence of ROS-induced fluorescence than cells within the confluent sides
(100 x image). Left frame shows fluorescence cells only, middle frame shows
all cells present (in phase contrast) and right frame is an overlay of left
and middle frames. Lower panel: 200x image showing greater detail of cells
migrating away from confluent side into open gap. Arrow points to direction
of the gap
| Time course of cell migration resulting in decreasing gap. Control (●)
and treatments with tempo (⊗) and N-acetyl cysteine (NAC; ∆)
are shown as the average ± S.D. of 8 replicate wells. Treatment samples
had significantly different slopes than controls (linear regression analysis,
95% confidence interval)
||Vertical migration of cells through FluorBlok transwell chambers
is inhibited by treatment with 2 mM tempol (Tmpo) and 2 mM N-acetyl cysteine
(NAC). Values represent the mean ± S.D. of cells per field of view
counted through a 20x objective. Five random areas were counted and averaged
per insert and 4 inserts per treatment condition were used. Both tempol
and NAC-treated cells migrated significantly slower (p<0.05; t-test)
compared with no treatment (no trt)
Untreated PC3M filled in the scratched area in five days; however tempo-treated
cells required 10 days to fill in the area and NAC-treated cells required a
13-day period (Fig. 2B). Thus, agents that enhance the breakdown
of ROS inhibit motility of PC3M.
In addition to the horizontal migration measurable with the scratch test, the
effect of inhibiting ROS on migration was also tested using transwell inserts
in a vertical migration assay. In this assay, inserts with 8 uM pores were inserted
into 24-well plates and cells which migrated through the pores from the upper
to lower chambers were enumerated. In this in vitro assay, both tempo
and NAC treatment inhibited migration by 52 and 76%, respectively (Fig.
2C). These data correlate well with the scratch results and further support
a role for ROS in directing cellular migration in this case through a porous
ROS could also promote growth in PC3M cells; however, we performed a proliferation assay and found that over a 6 day period, neither tempo nor NAC at the concentrations which inhibited motility significantly altered proliferation in PC3M over a 6 day period (data not shown).
Tempol and NAC Reduced Adhesion of PC3M on Selected Matrix Substrates
PC3M cells were pretreated overnight with tempol or NAC then assayed for
adhesion to vitronectin, fibronectin, Matrigel, which is a tumor-cell derived
basement membrane consisting primarily of laminin and collagen (Kleinman et
al., 1982) or type IV collagen (Fig. 3A). Control PC3M
cells showed greater levels of adhesion to fibronectin and Matrigel and adhesion
to these two extracellular matrix components was significantly reduced by both
tempol and NAC. In addition, NAC significantly reduced adhesion of PC3M to vitronectin.
No altered adhesion to type IV collagen was observed. The finding that tempol
and NAC inhibit adhesion as well as migration suggested the possibility that
ROS inhibition might also reduce invasion.
Tempol and NAC Inhibit in vitro Invasion
To assay invasion, transwell inserts were employed as above but in this
assay the inserts were coated with Matrigel. Tempo and NAC treatment significantly
reduced transwell invasion (Fig. 3B).
||Adhesion and invasion of prostate cancer cells is reduced
by treatment with 2 mM tempol and 2 mM NAC
|Prostate cancer cells were untreated (black bars) or treated
with tempol (white bars) or NAC (grey bars) and plated into matrix-coated
wells in a 96-well plate. Following a one-hour period for attachment, the
cells were detached and counted as described in Methods. Data is expressed
as the number of adherent cells; error bars show the SD from quadruplicate
wells. * indicates a significant difference compared to the untreated cells
for each matrix condition. Data were analyzed using one-way ANOVA
| In vitro invasion of PC3M cells is inhibited by 2 mM tempol and
2 mM NAC treatment. Cells plated in Matrigel-coated Fluoroblok inserts invaded
through the extracellular matrix to the lower surface of the transwell membrane.
Values calculated as described in 2C. Both tempol and NAC treatments significantly
inhibited invasion (p<0.05; t-test)
E. Levels of ROS Metabolizing Enzymes May Be Altered as a Function of Cell
To assess the activity of major degrading enzymes for ROS, activities were
measured for the enzymes superoxide dismutase (SOD) and catalase in low vs.
high density PC3M cells. In addition, the ratio of reduced to oxidized glutathione
was compared. These assays revealed that catalase activity in low density cells
was approximately one-half the activity in high density cells (Table
1). Neither SOD activity not the glutathione ratio varied significantly
||Comparison of ROS scavenging systems in PC3M cells grown to
low or high densities shows that catalase activity is reduced at low density.
Catalase and superoxide dismutase (SOD) activities are expressed as units/mg
Cancer metastasis is a complex process involving tumor cell separation from the primary tumor mass, extracellular matrix proteolysis, migration, motility, altered adhesion, and angiogenesis (Kawaguchi, 2005). It has been recently suggested that ROS could play important roles in growth and angiogenesis (Lim et al., 2005); however, in this study, our objective was to determine if ROS is associated with invasion-associated characteristics such as low density, motility, altered adhesion and in vitro invasion. Present results demonstrate that ROS are related to low cell density and are necessary for maximal directional motility, adhesion and invasion. These results suggest that prostate cancer cells may induce oxygen metabolites during the invasive process. These ROS could be by-products of increased energy production as Chowdhury et al. (2005) have previously shown that malignant prostate cells are more glycolytic than normal epithelia. Previous reports have indicated that ROS can activate several signaling pathways including the PI-3-kinase (PI3K) and Akt pathway in DU145 prostate cancer cells (Gao et al., 2004), extracellular signal-regulated kinases (Erk)-½ in gastric cancer (Kim et al., 2005a) and oxidant-sensitive transcription factors AP-1, CREB, and nuclear respiratory factor 1 (Felty et al., 2005) in breast cancer cell lines. Activation of these pathways is implicated in cancer metastasis (Poser and Bosserhoff, 2004; Kim et al., 2005a,b) and therefore the induced ROS in individual low density cells may contribute to development of a more invasive phenotype.
In our adhesion assay, we found that inhibition of ROS reduces adhesion to fibronectin and Matrigel basement membrane but this correlation did not extend not to type IV collagen. This suggests that ROS may selectively alter some components responsible for attachment. Whether these observations extend into the in vivo environment remains to be tested. Our observations from the scratch test indicate that only a portion of migrating cells intensely oxidized the indicator fluorochrome. This observation leads us to hypothesize that ROS upregulation during invasion and/or metastasis is a transient event and may not be associated with later stages of metastasis or with tumor establishment and high levels of homotypic adhesion.
The present findings are unique in that they suggest density is a key element in the induction of ROS in specific cells which are more motile and invasive. However, our data supports previous findings that have associated ROS with other invasion characteristics (Lim et al., 2005; Shariftabrizi et al., 2005; Gao et al., 2004; Chiarugi, 2003).
In order to further clarify the role of ROS in prostate cancer invasion and metastasis, it is necessary to elucidate specific pathways linked to migration and adhesion which are altered by the increased ROS. These pathways could be signaling pathways such as FAK phosphorylation (Chiarugi, 2003), modification of other growth factor pathways, integrins or matrix proteases.
This work was supported by funding through the LSU SVM CORP program and by the Louisiana Board of Regents LEQSF-RD-A-10 grant to IS.