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Endocrinological Oncology |
Departments of Diabetes, Endocrinology and Metabolism, (R.N., W.B., J.G., J.N.) Medical Oncology (R.E.) and Anatomic Pathology (S.W.), City of Hope National Medical Center, Duarte, California 91010
Address all correspondence and requests for reprints to: Jerry Nadler, M.D., Department of Diabetes and Endocrinology, City of Hope National Medical Center, 1500 East Duarte Road, Duarte, California 91010. E-mail: jnadler{at}smtplink.coh.org
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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LO products, such as the hydroxyeicosatetraenoic acids (HETEs), have been shown to have actions highly relevant to cellular growth and migration (3). They have significant mitogenic and chemotactic effects and also can stimulate the expression of several oncogenes (4, 5, 6, 7). The 12-LO product, 12(S)-HETE) has been shown to play a role in the growth-promoting effects of angiotensin II in vascular smooth muscle and adrenal cells (8, 9). Further, 12(S)-HETE has been shown to play a key role in mediating several major steps of the process of hematogenous metastasis of cancer cells (10, 11). Moreover, studies suggest that the biosynthesis of 12(S)-HETE by tumor cells is a determinant of their metastatic potential (12). The LO products of linoleic acid also can potentiate the mitogenic effects of epidermal growth factor (EGF) (13, 14), and linoleic acid can stimulate the growth of MCF-7 breast cancer cells (15, 16). However, few studies have examined the presence and regulation of the LO pathway in human breast cancer cells and tissues.
There are two major isoforms of 12-LOs, namely a platelet type and a leukocyte type (17, 18, 19). The platelet type of 12-LO has been cloned from human platelets and the megakaryocytic cell line, HEL (20, 21). The leukocyte type of 12-LO has been detected in porcine leukocytes (22), pituitary (23), vascular smooth muscle cells (24) and also in human adrenal glomerulosa cells (25), human monocytes, endothelial, and vascular smooth muscle cells (26). The porcine leukocyte 12-LO is only 65% homologous to the human platelet 12-LO (20, 21, 22), whereas it is 87% homologous to human 15-LO (22, 27). The two distinct 12-LO complementary DNAs (cDNAs) recently have been cloned from the same species, namely the mouse (28). Platelet 12-LO differs from leukocyte 12-LO in substrate specificity. The former is much less active with C18 fatty acids, such as linoleic acid, in comparison with arachidonic acid, whereas leukocyte 12-LO has broader substrate specificity, reacting with C18 and C22 unsaturated fatty acids as efficiently as with arachidonic acid (17).
In the present studies, we have examined whether the leukocyte-type 12-LO expression is upregulated in breast cancer cells and tissue sections and also whether EGF, a breast cell growth factor, can induce LO activity and expression in breast cancer cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Normal and malignant breast tissue was obtained from medically indicated surgical procedures. Use of discard human tissue samples from breast biopsies and mastectomies was approved by the City of Hope Institutional Review Board. Frozen tissue samples from primary breast tumors and uninvolved breast tissue from the same patients were processed for RNA extraction. The tumor samples included samples from five infiltrating ductal carcinomas and one highly metastatic carcinoma. Slides cut from various breast sections also were used for evaluating 12-LO protein cellular localization by immunohistochemistry.
Cell culture
MCF-7 breast cancer cell line was used in most of the studies. This and other breast cancer cell lines were obtained from ATCC (Rockville, MD). In some of the studies, we used other breast cancer cell lines (COH-BR1, MDA-MB-231, and T47D), as well as primary cultures of a normal breast epithelial cell (specimen 161, batch AC113, from Dr. Martha R. Stampfer of the University of California, Berkeley). We also used an immortal nontumorigenic breast epithelial cell line, MCF-10F. The MCF-10F line was obtained from ATCC. The COH-BR1 cell line (which is estrogen receptor negative) was developed at the City of Hope from malignant pleural effusions obtained as discard material from a medically necessitated procedure (29). All the breast cancer cell lines were maintained in DMEM containing 5% FCS, whereas the MCF-10F and normal AC113 cells were maintained in the medium MCDB-170-SFS, as described by Hammond et al. (30).
Measurement of the LO product 12-HETE
Nearly confluent MCF-7 cells in 100-mm dishes were serum depleted by placing in DMEM/HEPES, containing 0.2% BSA and 0.4% FCS, for 24 h. Just before the experiment, the cells were placed in media containing 0.2% BSA, preincubated for 20 min at 37 C, and then treated with or without EGF (human recombinant, Gibco BRL, Gaithesburg, MD) for a further period of 4 h. The reaction was terminated by cooling on ice. The HETEs in the supernatants and cell pellets (cell-associated) were extracted as described earlier (24). 12-HETE in the cell extracts was quantitated by a specific RIA (24). The 12-HETE RIA is specific for 12(S)-HETE, with less than 0.1% crossreactivity with 12(R)-HETE. The identity of the HETE was confirmed by comigration with authentic cold 12(S)-HETE using our gradient reverse-phase high-performance liquid chromatography (HPLC) system (34).
Measurement of LO activity
MCF-7 cells in 100-mm dishes were serum depleted for 24 h and then treated with EGF for 4 h. Cells were then harvested, washed, suspended in 1 mL Tris-HCl buffer (25 mM, pH 7.7) (about 2 x 106 cells), and then sonicated on ice. 12-LO activity in the sonicates was estimated as described earlier (24).
Incubations for 12-LO protein or messenger RNA (mRNA) expression
MCF-7 cells (about 80% confluent) in 100-mm dishes were serum depleted for 24 h by placing them in DMEM/HEPES and 0.2% BSA + 0.4% FCS. The above medium was then freshly added alone or with EGF and the cells incubated at 37 C. At the end of the incubation time period, the cells were processed for Western blotting or RNA extraction, as described below.
Electrophoresis and Western immunoblotting
Washed cell pellets were lysed in lysis buffer, and lysates were centrifuged at 5000 rpm for 10 min and supernatants subjected to electrophoresis and Western Blotting, as described earlier (24, 25). Detection was by the Western Light Chemiluminescent system (Tropix Inc., Bedford, MA). Authentic porcine leukocyte 12-LO protein was obtained from Oxford Biomedical Research Inc. (Oxford, MI), and used as a positive control. Western blots were quantitated using a computerized video densitometer (Applied Imaging Lynx DNA vision, Santa Clara, CA) and values expressed as arbitrary optical density units.
In some experiments, in order to test for the presence of human platelet 12-LO, a polyclonal antibody to human platelet 12-LO (Oxford Biomedical) was used at a dilution of 1:400. At this dilution, this platelet 12-LO antibody will not cross-react with porcine 12-LO.
cDNAs
pUC19 plasmid, containing the cDNA for porcine leukocyte 12-LO (22), was a generous gift from Dr. T. Yoshimoto, Tokushima, Japan. Recombinant Bluescript plasmid containing the cDNA for human reticulocyte 15-LO was kindly provided by Dr. E. Sigal (University of California, San Francisco, CA). Bluescript plasmid, containing the cDNA for human platelet 12-LO, was kindly provided by Prof. Bengt Samuelsson (Karolinska Institute, Stockholm, Sweden) (21). The full-length 15-LO cDNA, porcine leukocyte 12-LO cDNA, and platelet 12-LO cDNA were prepared by EcoRI, SalI, and NotI digestion of the plasmids, respectively.
Oligonucleotide primers and probes for PCR
All the oligonucleotides, including human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) oligonucleotides, were synthesized on an Applied Biosystems (Foster City, CA) DNA synthesizer in the Beckmann Research Center (City of Hope) nucleotide synthesis facility and were purified by PAGE. The sequences of oligonucleotides are as shown earlier (24, 25) and were designed based on known gene sequences (22, 27, 31) and selected from regions displaying most divergence between porcine 12-LO and human 15-LO sequences (20), caused by their close homology.
Amplification of reverse transcribed RNA using RT-PCR
Normal and cancer involved breast tissue samples or MCF-7 cells that had been treated with or without EGF were subjected to total RNA extraction by phase partition using the guanidium thiocyanate-phenol-chloroform extraction method with RNA STAT 60 (Tel Test "B" Inc., Friendswood, TX). Total RNA (1 µg) was mixed with the PCR buffer (10 mmol/L Tris-HCl, pH 8.3, 50 mmol/L KCl, 1.5 mmol/L MgCl2, 0.001% gelatin), 200 µmol/L of each of the four deoxynucleotide trisphosphates, 25 pmol each of 5' and 3' primers (24, 25), 2 U RNasin (Promega Corp., Madison, WI), 2 U avian myeloblastosis virus RT (20 U/µL, Life Sciences, St. Petersburg, FL), and 2.5 U Taq polymerase (Perkin Elmer Cetus, Norwalk, CT), in a final vol of 50 µL. In all reactions, 5 pmol of each 5' and 3' primers of GAPDH were added as an internal standard to control for RNA quantity and amplification efficiency. We have standardized conditions and used a number of cycles such that the amplification of both 12-LO and GAPDH are each in the linear range of amplification, as also reported earlier (24, 25). The samples were placed in a DNA thermal cycler (Perkin Elmer Model 480) at 37 C for 15 min for the RT reaction to proceed. Then conditions used for PCR were a denaturation step at 94 C for 1 min, annealing at 50 C for 2 mins., and extension at 72 C for 2 min for 25–30 cycles. Blank reactions with no RNA template or with no RT were carried out through the RT and PCR steps. Amplifications of the appropriate cDNAs were used as positive controls for PCR. RNA from the erythroleukemia cell line, HEL, which express only platelet 12-LO (20), were run as negative controls for leukocyte-type 12-LO.
The PCR products were subjected to Southern blotting and hybridization to detect 12-LO, as described earlier (24, 25). The washing and hybridization conditions were developed to distinguish between the PCR products of human 15-LO from those of porcine leukocyte 12-LO (25) caused by their close homology. Using these conditions, we have shown that the 12-LO primers do not amplify 15-LO mRNA and vice versa (25). After autoradiography, blots were quantitated using a computerized video densitometer (Applied Imaging Lynx).
Immunohistochemistry
The immunohistochemical methods were carried out using a previously published technique with modifications (32). The primary (leukocyte 12-LO) antibody was used at a dilution of 1:1000. Briefly, 4-micron paraffin-embedded tissue sections were mounted on Probe-on slides (Ventana Medical Systems, Tucson, AZ) and dried overnight in a 56-C oven, deparaffinized in xylene, and rehydrated in graduated alcohol to distilled water. The slides were loaded into a Techmate Slide holder and placed into 0.1 mol/L citrate buffer solution for heat-induced epitope retrieval (33) using a household Black and Decker (Shelton, CT) steamer (model no. HS90). The slides were steamed in 0.1 mol/L citrate buffer for 20 min and then allowed to cool for 5 min. After first and second antibody treatments, slides were stained using a modified ABC technique, using DAB as chromogen, and counterstained using Mayer’s hematoxylin. Staining was performed using a Bioteck Techmate 1000 Immunostainer (Teckmate, Santa Barbara, CA) with Biotek Solutions and ABC detection system (Teckmate). Parallel controls were run for each specimen without primary antibody.
Growth curves
MCF-7 cells were plated in 6-well dishes in DMEM containing 10% FCS. After 48 h, they were treated with LO or CO inhibitors (all obtained from BIOMOL Research, Plymouth Meeting, PA) or the corresponding vehicle (0.1% dimethyl sulfoxide). Cell counts were obtained at 48 h intervals after trypsinization. Fresh medium and inhibitors were replaced every 48 h.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether 12-LO mRNA expression was altered in breast
cancer tissues, we screened 6 sets of uninvolved and cancer-involved
breast tissue samples from 6 patients for the presence of the
leukocyte-type 12-LO mRNA. Total RNA was obtained from these samples,
and we used RT-PCR to detect 12-LO mRNA levels, owing to its low levels
in these tissues. Figure 1
, a and b, shows Southern
blots run with the RT-PCR products from the 12 samples obtained from
these 6 patients. Hybridization was performed with a
[32P]-labeled porcine leukocyte 12-LO oligonucleotide
probe (upper panels of both a and b). The size of the
expected PCR product is 333 bp. The positive control, namely leukocyte
12-LO cDNA, is seen at the far right. It is clearly seen
that in each patient, the cancerous section had a much higher level of
12-LO mRNA expression than the corresponding normal section. In fact,
in patients 4–6, 12-LO mRNA was barely expressed in the normal
sections. After correction for amplification of the internal control,
GAPDH mRNA (PCR product 284 bp), densitometric analysis (shown in Table 1) revealed 3- to 30-fold greater 12-LO mRNA expression
in the cancer sections than in the corresponding uninvolved sections
from the same patients. These results suggest that malignant breast
tissues express a much higher level of the 12-LO mRNA in
vivo, compared with matched uninvolved tissues.
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Human 15-LO mRNA expression in breast tissue samples
Because the human 15-LO and the leukocyte-type 12-LO are very
homologous, we examined the expression of 15-LO mRNA in the same
patient tissue samples as above. We used a specific RT-PCR approach
that distinguishes between 15-LO and the leukocyte-type 12-LO (25).
Fig. 2 is a Southern blot of the amplified products
obtained by RT-PCR to examine 15-LO mRNA expression. RNA from the
matched normal and cancer tissue sections from the same patients 1, 2,
4, 5, and 6 were studied. Patient 3 was not be studied because of
paucity of material. The positive control, 15-LO cDNA, is seen on the
far right. The size of the 15-LO PCR product is 333 bp. The
results demonstrate expression of 15-LO mRNA in human breast tissue and
cancer. However, 15-LO mRNA expression was enhanced in the
cancer-involved section in only two of the samples. Furthermore, in
the other three patient samples, the normal tissue had much greater
15-LO mRNA expression. Densitometric representation of the data is seen
in Table 1
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Because tissue samples contain a variety of cell types, we also
examined breast cell lines for the presence of the leukocyte-type 12-LO
mRNA and compared its expression in normal vs. cancer cell
lines. Figure 3A shows a Southern blot of the RT-PCR
amplified products from total RNA from two breast cancer cell lines
(MCF-7 and COH-BR1), as well as an immortal, nontumorigenic breast
epithelial cell line (MCF-10F). The results clearly show that there is
very little basal expression of 12-LO mRNA (333-bp PCR product) in
MCF-10F cells (Fig. 3A). However, distinct expression of 12-LO was seen
in the two cancer cell lines, MCF-7 and COH-BR1 (7- and 11-fold greater
than the MCF-10F cells). The positive control for PCR, 12-LO cDNA
amplification, is seen in the far right. GAPDH mRNA
amplification (284-bp PCR product), shown in the lower panel
of Fig. 3A, indicates that the low 12-LO mRNA levels in the MCF-10F
cells is not caused by paucity of template. These results suggest that
breast cancer cell lines, such as MCF-7 and COH-Br1, have a much higher
level of expression of 12-LO mRNA, compared with the control cell line,
MCF-10F.
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show that all the four breast cancer cell
lines display a clear band around 75 kD similar to the band seen with
human vascular smooth muscle cells on lane 5. Authentic porcine
leukocyte 12-LO is in lane 1. The human 12-LO seems to migrate at a
slightly higher molecular size than porcine 12-LO. In contrast, the
normal breast epithelial cell line does not show this 12-LO band with
or without 24-h EGF treatment (lanes 6 and 7). The effect of EGF on cell-associated 12(S)-HETE levels
To evaluate whether a potent breast epithelial cell growth factor
can affect the LO pathway, we examined whether EGF can increase the
formation of the 12-LO product, 12(S)-HETE, in MCF-7 breast cancer
cells. A 4-h treatment of the cells with EGF did not affect the levels
of released 12(S)-HETE. In contrast, this treatment with EGF led to a
dose-dependent increase in the levels of cell-associated 12(S)-HETE, as
seen in Fig. 4. Thus, EGF from 25–100 ng/mL led to
significant increase in the levels of cell-associated 12(S)-HETE.
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We also examined whether treatment of the MCF-7 cells with EGF
leads to an increase in intracellular 12-LO enzyme activity, as
assessed by the conversion of substrate arachidonic acid to 12(S)-HETE
by cell sonicates. Figure 5 shows the HPLC tracings of
extracts of sonicates from MCF-7 cells treated with or without EGF for
24 h. The first panel depicts the retention times of
the authentic cold standards 12(S)- and 15(S)-HETE. The second
panel reveals the HPLC tracing of 12-LO activity in control cells,
where a distinct peak with the same retention time as 12-HETE is seen
(arrow). Further, the next two panels show that
treatment with EGF at 25 and 50 ng/mL led to an increase in the height
of the 12-HETE peak (1.4- and 2-fold, respectively), thus indicating
that EGF can increase 12-LO enzyme activity in these cells. The
identity of the 12-HETE peak in the HPLC tracings was confirmed by
comigration with authentic cold 12(S)-HETE, as well as by observing a
quantitative increase in the height of the 12-HETE peak when coinjected
with a known amount of authentic cold 12(S)-HETE.
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We next examined the effect of EGF on leukocyte-type 12-LO protein
expression in MCF-7 cells. 12-LO protein in lysates of cells treated
with or without EGF was identified by immunoblotting using a specific
polyclonal peptide antibody to porcine leukocyte 12-LO. We previously
have shown that this antibody can detect the leukocyte-type 12-LO in
human tissues and cells (25, 26). Figure 6 shows that a
36-h treatment with EGF leads to a marked increase in levels of the
12-LO protein (approximately 75 kDa). This increase was seen, beginning
with 10 ng, with a maximal effect at 25 ng/mL EGF. The bar graph in
Fig. 6 shows the densitometric quantitation of the blot and reveals
that EGF can lead to a 2- to 3-fold increase in 12-LO expression.
Authentic porcine leukocyte 12-LO is shown in the far left
lane. It is noted that the 12-LO in these human MCF-7 cells
appears at a slightly higher molecular mass than the porcine 12-LO. A
similar band, however, also was observed when we used another antibody
directed against a different peptide derived from the N-terminal end of
porcine leukocyte 12-LO (amino acids 39–55) (results not shown).
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shows that lysates from
HEL cells, which are a positive control for human platelet 12-LO (20),
show clear expression of platelet 12-LO. In contrast, equal amounts of
lysate protein from MCF-7 cells alone, or treated with 25–100 ng/mL
EGF, loaded in the adjoining lanes, do not show evidence for
the presence of platelet 12-LO. The last two lanes in Fig. 7
show that the antibody at the concentration used does not cross-react
with authentic porcine 12-LO or with human 15-LO.
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Sections of breast cancer tissue from four of the six patients
studied were examined for the presence of leukocyte-type 12-LO, by
immunohistochemical methods. The peptide leukocyte 12-LO antibody was
used at 1:1000. Figure 8 shows the results from a
representative stained section from a moderately differentiated ductal
adenocarcinoma. The section shows clear, strong granular cytoplasmic
staining for 12-LO in the tumor epithelial cells seen in the
center. In addition, we could see scattered staining in some
of the surrounding lymphocytes, histiocytes, vascular endothelium, and
also in benign ductal epithelial cells in all the sections studied. The
negative controls without the primary antibody showed no staining (not
shown).
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To evaluate the potential functional significance of altered 12-LO
expression in the breast cancer cells, we examined the effect of two
specific, structurally dissimilar 12-LO inhibitors,
cinnamyl-3,4-dihydroxy-
-cyanocinnamate (CDC) and baicalein, on the
proliferative rates of MCF-7 cells. The results seen in Fig. 9 show that both LO inhibitors led to a marked
inhibition of the serum-induced growth of these cells. Because both CDC
and baicalein may also block the 5-LO pathway, we also checked the
effect of a highly specific 5-LO inhibitor, AA-861. Fig. 9 shows that
although this 5-LO inhibitor does have significant inhibitory effects
on the proliferation of the MCF-7 cells, it is not as potent as CDC or
baicalein. To evaluate the specificity of these effects, we also
compared the effect of a CO inhibitor, ibuprofen. Fig. 9 shows that
ibuprofen, at the same concentration as CDC and baicalein, had no
significant effect on the proliferation of the cells. These results
indicate that the LO pathway may mediate, at least in part, the growth
of breast cancer cells. However, 12-HETE may not be the only LO product
involved in breast cancer, and other LO products generated by the 5-LO
or other pathways also may play a role.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The immunohistochemical data revealed strong staining in cancer cells with a leukocyte-type 12-LO antibody, indicating the presence of 12-LO in these cancer cells. This does not completely rule out some 15-LO staining caused by the high homology with leukocyte 12-LO, and this may explain some of the staining in the benign ductal tissue. The PCR method used was more specific and can specifically distinguish between human 15-LO and the leukocyte 12-LO. Using PCR, we found a clear difference between 15-LO vs. leukocyte 12-LO mRNA expression in cancer involved vs. uninvolved samples from patients.
Evidence suggests that tumor cells and several normal cells have LO activity (3, 34), and both arachidonic acid and linoleic acids are converted to LO products, such as HETEs and hydroxyoctadecadienoic acids (HODEs). The 12-LO product, 12(S)-HETE, has been shown to play an important role in the metastatic process (10, 11). 12(S)-HETE mediates the adhesion of tumor cells to the subendothelial matrix after endothelial retraction by a protein kinase C-dependent process (34, 35). LO products, such as 12- and 15-HETE, also have mitogenic effects on endothelial cells (4). Liu et al. have demonstrated that 12-HETE is the predominant arachidonic acid metabolite produced by highly metastatic tumor cells. Furthermore, these highly metastatic cells synthesize much greater amounts of 12-HETE than the low metastatic tumor cells (12). Thus, an increased concentration of 12-HETE, produced by activated platelets, the tumor cells themselves, leukocytes, or by vascular cells, could facilitate the proliferative and metastatic processes. In the present study, we did not find clear evidence for a platelet-type 12-LO protein in the MCF-7 breast cancer cells. However, our studies do not rule out the possibility that a platelet-type of 12-LO also contributes to 12(S)-HETE formation in tissue samples and in the local environment of tumor cells.
LO metabolites of arachidonic acid have been reported to mediate tumor necrosis factor-induced protooncogene c-fos expression (7). In addition, EGF-induced mitogenic activity has been linked to the formation of LO products of linoleic acid, the HODEs (13, 14). Linoleic acid metabolism enhances the proliferative response in mouse mammary epithelial cells and in human breast epithelial cells (36, 37). The direct growth effects of linoleic acid, however, seemed more visible with the ER-negative cell line MDA-MB-231 than with the ER-positive MCF-7 cells (15). LO products, rather than CO products, were found to play a major role in linoleic acid-stimulated growth of mouse mammary tumor cell line (38). Thus, a growing body of evidence suggests that specific metabolites of arachidonic and/or linoleic acid serve as central elements in signal pathways necessary for cell mitogenesis, as induced by growth factors or oncogenic transformation. Our present results, implicating a role for the 12-LO pathway in the development and progression of breast cancer, therefore, yield new information on the processes leading to neoplastic growth.
In A431 epidermoid carcinoma cells, EGF could induce platelet 12-LO mRNA expression (39). In the present studies, we have shown that EGF also can induce a leukocyte-type 12-LO in MCF-7 breast cancer cells. We recently have demonstrated the presence of a leukocyte-type 12-LO in human adrenal monocytes (25), vascular smooth muscle cells, and endothelial cells (26). Human vascular smooth muscle cell 12-LO expression was increased by treatment with angiotensin II (26), whereas human monocyte 15-LO was induced by interleukin-4 and interleukin-13 (40, 41). Furthermore, we showed that porcine vascular smooth muscle cells express a leukocyte-type 12-LO, the activity and expression of which was increased by angiotensin II, as well as by high-glucose culture conditions (24). Thus, increased leukocyte-type 12-LO activity and expression may play a role in the growth-promoting effects of these factors (8).
In this study, we have not examined the mechanisms by which LO products mediate breast cancer cell growth, but evidence indicates that they can initiate several growth-related signaling events, such as activation of oncogenes, protein kinase C, and mitogen-activated protein kinases (42). The present results suggest that human breast cancer tissues and cell lines show increased 12-LO activity and expression, which may play a key role in breast cancer growth and/or progression.
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Acknowledgments |
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Footnotes |
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Received June 26, 1996.
Revised November 25, 1996.
Revised February 14, 1997.
Accepted February 21, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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