Dietary fatty acids regulate the activation status of Her-2/neu (c-erbB-2) oncogene in breast cancer cells
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Ankara University School of Medicine, Department of Medical Oncology
,
Ankara
,
Turkey (
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Research from several sources provides strong evidence that
dietary or exogenously derived fatty acids (FAs) may play an
important role in the etiology, evolution and/or progression of
breast cancer [1]. However, the type of individual FAs in a
diet, rather than the amount of total dietary fat, may be more
important in breast cancer disease. In this regard,
epidemiological, experimental and mechanistic data implicate v-9 oleic
acid (OA; 18:1n-9), the main FA of olive oil, a-linolenic acid
(ALA; 18:3n-3), the v-3 FA of vegetable oils, and
eicosapentaneoic (EPA; 22:5n-3) and docosahexaenoic (DHA; 22:6n-3)
FAs, the two main v-3 FAs of fish oils, as inhibitors of the
development and progression of human breast cancer, and v-6
FAs such as linoleic acid (LA; 18:2n-6), the main FA of corn
and sunflower oils, as stimulators of the disease. Moreover,
one of the most interesting yet controversial dietary
approaches has been the possible role of dietary FAs in
treating breast cancer. However, little is know about the ultimate
mechanisms underlying FA-regulated breast cancer risk and
progression. Interestingly, we previously reported that
exogenous supplementation with FAs such as g-linolenic acid (GLA;
18:3n-6), an essential FA found in the plant-seed oils of
evening primrose, blackcurrant and borage oils, synergically
sensitizes cultured breast cancer cells to anti-mitotic drugs such
as paclitaxel (Taxole) [2], vinorelbine (Navelbinee) [3] and
docetaxel (Taxoteree) [4]. Moreover, this GLA-induced
sensitization was also synergic with the anti-estrogens tamoxifen
and ICI 182,780 (Faslodexe) [5]. Since the human Her-2/neu
(c-erb B-2) oncogene and its p185Her-2/neu orphan receptor
oncoprotein, which are overexpressed in 25 30% of invasive
breast cancers, have been associated with cytotoxic and
endocrine therapy resistance [6], we recently envisioned that
FA-induced breast cancer chemosensitization may involve
regulation of Her-2/neu-dependent signaling.
In the last few years it has become clearer that the
activation status of Her-2/neu, and not just its overexpression, is a
crucial event that determines both the aggressive biological
behavior of breast carcinomas and the breast cancer response
to chemotherapy, anti-estrogens and the anti-Her-2/neu
antibody trastuzumab (Herceptine) [7, 8]. Although the
mechanism of activation of the Her-2/neu oncoprotein is not
completely understood, in vitro and in vivo Her-2/neu
activation has been demonstrated to occur as a consequence of
proteolytic cleavage of its extracellular domain (ECD),
thereby resulting in the production of truncated
membranebound fragment with kinase activity, a key event for
downstream signaling. In this regard, we recently reported that
Her-2/neu ECD expression correlates with a diminished
efficacy of the combination chemotherapy of biweekly
gemcitabine plus paclitaxel [9]. This study validated our previous
results, showing that both the probability of obtaining a
complete response and the duration of clinical response to a
paclitaxel doxorubicin chemotherapy regimen were significantly
lower and shorter, respectively, in metastatic breast cancer
patients with increased Her-2/neu ECD concentrations
compared with the cases with non-increased concentrations [10].
The present investigation was performed to evaluate the
effects of exogenous supplementation with dietary FAs on
Her-2/neu ECD concentrations in cultured breast cancer cells.
First, the Oncogene Science Her-2/neu Microtiter ELISA
(Cambridge, MA, USA) was used to compare the baseline
expression of Her-2/neu ECD in BT-474, SK-Br3, MCF-7 and
MDA-MB-231 human breast cancer cell lines. This
sandwichtype enzyme immunoassay utilizes two monoclonal
antibodies, NB-3 and TA-1, that recognize the extracellular,
ligand-binding domain of the Her-2/neu oncoprotein, and
quantifies the Her-2/neu ECD in cell cultures. As expected,
very high levels of Her-2/neu ECD [expressed as human Neu
units (HNU) per mg of protein] were found in BT-474 and
SK-Br3 breast cancer cells (10512 and 9511 HNU per mg
protein, respectively), whereas MCF-7 (1.6 HNU/mg protein)
and MDA-MB-231 (0.5 HNU/mg protein) cells contained
physiological and low levels of Her-2/neu ECD, respectively.
Thus, Her-2/neu ECD levels in Her-2/neu-overexpressing
SKBr3 and BT-474 breast cancer cells were 200-fold higher
than in Her-2/neu-negative MDA-MB-231 cells.
To assess the effects of FAs on Her-2/neu ECD
concentration, SK-Br3 and BT-474 cells, after a 24 h starvation
period in media without serum but with 0.5% FA-free bovine
serum albumin (BSA), were incubated for 48 h with 0.5%
BSA FA-free BSA (controls) or various concentrations (1.25,
2.5, 5 and 10 mM) of BSA-bound FAs (Table 1). Her-2/neu
ECD levels in SK-Br3 cells were slightly decreased by 24% at
10 mM ALA, while Her-2/neu ECD expression dramatically
decreased by 53% with 10 mM ALA in BT-474 cells. Cultur (...truncated)