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. 2018 Jul 25;41(5):505–516. doi: 10.1007/s13402-018-0387-3

Inositol-C2-PAF acts as a biological response modifier and antagonizes cancer-relevant processes in mammary carcinoma cells

Carsten Pelz 1,#, Sonja Häckel 1,2,#, Geo Semini 1,3, Sandra Schrötter 1,4, Willem Bintig 1, Sebastian Stricker 1, Gudrun Mrawietz 1, Andreas Klein 1, Lothar Lucka 1, Vadim Shmanai 5, Britta Eickholt 1, Annette Hildmann 1,6,#, Kerstin Danker 1,✉,#
PMCID: PMC12995239  PMID: 30047091

Abstract

Purpose

Previous studies have identified alkyl-phospholipids as promising compounds for cancer therapy by targeting constituents of the cell membrane and different signaling pathways. We previously showed that the alkylphospholipid Inositol-C2-PAF inhibits the proliferation and migration of immortalized keratinocytes and the squamous carcinoma-derived cell line SCC-25. Here, we investigated the effect of this compound on growth and motility as well as its mode of action in mammary carcinoma-derived cell lines.

Methods

Using BrdU incorporation and haptotactic cell migration assays, we assessed the effects of Inositol-C2-PAF on MCF-7 and MBA-MB-231 cell proliferation and migration. The phosphorylation status of signaling molecules was investigated by Western blotting as well as indirect immunofluorescence analysis and capillary isoelectric focusing.

Results

We found that Inositol-C2-PAF inhibited the growth as well as the migration in MCF-7 and MBA-MB-231 cells. Furthermore, we found that this compound inhibited phosphorylation of the protein kinase Akt at serine residue 473, but had no impact on phosphorylation at threonine 308. Phosphorylation of other kinases, such as Erk1/2, FAK and Src, which are targeted by Inositol-C2-PAF in other cells, remained unaffected by the compound in the mammary carcinoma-derived cell lines tested. In MCF-7 cells, we found that IGF-1-induced growth, as well as phosphorylation of AktS473, mTOR and the tumor suppressor pRB, was inhibited in the presence of Inositol-C2-PAF. Moreover, we found that in these cells IGF-1 had no impact on migration and did not seem to be linked to full Akt activity. Therefore, MCF-7 cell migration appears to be inhibited by Ino-C2-PAF in an Akt-independent manner.

Conclusion

The antagonistic effects of Inositol-C2-PAF on cell migration and proliferation are indicative for its potential for breast cancer therapy, alone or in combination with other cytostatic drugs.

Electronic supplementary material

The online version of this article (10.1007/s13402-018-0387-3) contains supplementary material, which is available to authorized users.

Keywords: Breast cancer, Inositol-C2-PAF, Proliferation, Migration, MDA-MB-231, MCF-7, Akt phosphorylation, IGF-1

Introduction

Breast cancer is one of the most common malignancies in women and comprises 23% of total new cancer cases and 14% of total cancer deaths worldwide [1]. Significant advances have been made in the treatment of most primary breast cancers [2, 3]. However, due to undesirable side effects and drug resistance, there is still an unmet need for novel chemotherapeutic compounds. Additionally, some mammary tumors, such as triple-negative or inflammatory mammary carcinomas, with high rates of metastasis and a poor prognosis, remain challenging regarding successful therapy. In this respect, the motility of cancer cells that leads to dissemination from the primary tumor to a distant organ by sequential migration steps plays a key role in tumor progression and often contributes to the highly detrimental effects of the tumor [4, 5].

Inositol-C2-platelet activating factor (Ino-C2-PAF) is a synthetic alkylphospholipid (APL) that effectively inhibits the proliferation of different hyperproliferative cells [6, 7], whereas it exhibits a low cytotoxicity in normal cells and tissues in vitro as well as in vivo [8, 9]. In lymphoma-derived cell lines, Ino-C2-PAF induces apoptosis [6], and it inhibits the motility of immortal keratinocyte-derived HaCaT cells and squamous carcinoma-derived SCC-25 cells. These effects are accompanied by Ino-C2-PAF-dependent inhibition of phosphorylation of two key regulators of cell motility, i.e., focal adhesion kinase (FAK) and Src [7]. Using a microarray-based gene expression technology, we could demonstrate that, in addition to this direct effect on signaling molecules, Ino-C2-PAF alters the expression of various genes. Particularly, we found that Ino-C2-PAF can repress the expression of a group of targets implicated in inflammation and innate immune responses [10, 11], indicating a role as a biological response modifier.

In the present study, the impact of this compound on two different breast cancer-derived cell lines, MDA-MB-231 and MCF-7, which represent distinct pathological subtypes of breast cancer, was investigated. We show that Ino-C2-PAF inhibits the growth as well as the motility of both cell lines, even at concentrations clearly below the LC50. Additionally, we show that Ino-C2-PAF inhibits the phosphorylation of protein kinase Akt at serine residue 473, whereas the phosphorylation of this kinase at threonine residue 308, as well as phosphorylation of the protein kinases Erk1/2, FAK and Src, remains unaffected. We also show that the growth of MCF-7 cells, which is elicited by the known Akt activator IGF-1, is inhibited in the presence of Ino-C2-PAF. This correlates well with phosphorylation of Akt at serine residue 473. However, the migration of MCF-7 cells was not found to be affected by IGF-1. This indicates that the Ino-C2-PAF-dependent inhibition of cell migration in these cells is achieved by an Akt-independent mechanism.

Materials and methods

Cell cultures and compounds

MDA-MB-231 cells, which are derived from an aggressive breast cancer, are negative for the estrogen receptor (ER) and the progesterone receptor (PR), and exhibit no amplification of the human epidermal growth factor receptor 2 (HER2; triple negative). MCF-7 cells, which are derived from a highly differentiated breast cancer, are positive for ER, PR and low levels of HER2 [12]. Both cell lines were a kind gift of Göran Landberg (Sahlgrenska Cancer Center, University of Gothenburg, Gothenburg, Sweden). The cells were cultured in DMEM containing 25 mM glucose, penicillin/streptomycin (100 U ml−1), L-glutamine (440 mg l−1) heat-inactivated fetal calf serum (FCS; 5% for MDA-MB-231 and 10% for MCF-7) at 37 °C in a humidified incubator with 5% CO2. MCF-7 cells were harvested using phosphate buffered saline (PBS)/0.02% EDTA, whereas MDA-MB-231 cells were harvested using 0.05% trypsin/0.02% EDTA in PBS. For the experiments, both cell lines were stimulated in the presence of 2% FCS after reaching 70% confluence (unless stated otherwise). Therefore, the FCS concentration of MCF-7 cells was reduced to 5% overnight before stimulation. Immortal keratinocyte HaCaT cells were grown in RPMI-1640 medium supplemented with heat- inactivated fetal bovine serum (FBS; 10%), penicillin (100 U/ml), streptomycin (0.1 mg/ml) and L-glutamine (440 mg/l). The cells were harvested using trypsin and 0.02 mM EDTA. Two days prior to experimentation, the cells were adapted to defined keratinocyte serum-free medium with growth supplements (pituitary extract including insulin, epidermal growth factor (EGF) and fibroblast growth factor (FGF) (Gibco/BRL). The experiments were performed in defined keratinocyte serum-free medium (Gibco/BRL). Ino-C2-PAF was synthesized as described previously [8]. IGF-1 was purchased from Biomol (Hamburg, Germany) and was added at a concentration of 100 ng/ml−1 for the experiments.

Cell proliferation assay

A colorimetric immunoassay (cell proliferation ELISA with bromodeoxyuridine (BrdU); Roche Diagnostics, Mannheim, Germany) was used to detect replicating cells according to the manufacturer’s instructions. Briefly, cells were seeded in a 96-well microtiter plate at 2 × 104 cells per well and cultured for 24 h before stimulation. Subsequently, the cells were incubated with different concentrations of Ino-C2-PAF for 48 h. Twenty-four hours before measurement, 100 μM BrdU labeling solution was added to reach a final concentration of 10 μM BrdU per well. The colorimetric reaction was determined photometrically at 405 nm and subsequently quantified. Untreated cells were set at 100%.

Soft agar colony formation assay

The effects of Ino-C2-PAF on anchorage-independent cell growth were evaluated using a neoplastic transformation assay in soft agar. The assay was carried out as described by Gottschalk and Klein [13]. Briefly, MDA-MB-231 and MCF-7 cells (4 × 104 ml−1) were treated in 6-well tissue culture plates containing 1 ml 0.33% low melting agarose (SeaPlaque®GTG® Agarose, Lonza Cologne GmbH, Germany) supplemented with 5% FCS over solidified 0.5% agarose-containing DMEM/10% FCS in the presence of various concentrations of Ino-C2-PAF. MCF-7 cells were maintained in a 37 °C, 5% CO2 incubator for 6 days, after which cell colonies were scored using a Zeiss Axiovert 200 microscope with an Axiocam. MDA-MB-231 colonies were allowed to grow for 15 days and were subsequently scored. The size of the colonies was determined using Axiovision software (AxioVs40V; Zeiss, Jena, Germany). The size of the respective colonies grown in the absence of Ino-C2-PAF was set at 100%.

Cytotoxicity assay

The cytotoxic effect of Ino-C2-PAF was determined using a commercial cytotoxicity detection kit (LDH; Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. Briefly, cells (1.5 × 104 cells per well) were incubated in the presence of the indicated Ino-C2-PAF concentrations for 48 h or were left untreated. The colorimetric reaction was measured photometrically at 492 nm and was subsequently quantified. Untreated cells served as a negative control (0% cytotoxicity), whereas cells lysed with Triton X-100 (0.5%) served as a positive control and were set at 100% cytotoxicity.

Cell migration assay

The migration of MDA-MB-231 and MCF-7 cells was characterized using Transwell systems (Corning Inc., USA; 8.0 μm pore size). The lower surfaces of the membranes were coated with collagen I (20 μg ml−1; Sigma, Germany) at room temperature for 30 min and subsequently washed with PBS. Cells were harvested, washed and resuspended in serum-free medium. Next, 5 × 104 cells were added to the upper chamber of the Transwell system and allowed to migrate in the presence or absence of Ino-C2-PAF and/or IGF-1 (100 ng ml−1). The lower chamber contained medium with 10% FCS. After 22 h of incubation, the cells on the upper surface were removed, and migrated cells on the lower surface were fixed with 3% paraformaldehyde/0.025% saponin in PBS and stained with 0.1% crystal violet. The cells were counted using a 10 × 10 grid at 250 × magnification. Five fields per filter were counted and averaged.

Western blotting

For Western blotting, the cells were washed with PBS and then lysed in Bicine/CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) and sample diluent (1× dimethylsulfoxide (DMSO) inhibitor mix, 1× aqueous inhibitor mix) (all Protein Simple, Santa Clara, USA) on ice for 45 min. This lysis buffer was also used for the NanoPro100™ analyses. The lysed cells were centrifuged for 15 min at 4 °C at 11,000 g after which the supernatants were denatured by boiling in Laemmli’s sample buffer. The samples were separated under reducing conditions by 7.5% or 10% SDS-PAGE and blotted onto nitrocellulose membranes (Thermo Fisher, Rockford, USA). The membranes were subsequently blocked in 5% BSA/TBS/0.1% Tween-20 overnight and then incubated with the respective primary antibodies in blocking buffer overnight at 4 °C. The primary antibodies and the respective working concentrations used are listed in Table 1. After incubation with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies, the proteins were detected using a SuperSignal West Pico Chemiluminescent Substrate (Pierce, Thermo Fisher Scientific, Bonn, Germany). The signals were visualized using VersaDoc™ 4000 MP and QuantityOne® 4.6.5 software (BioRad Laboratories, Munich, Germany) and quantified using Image J 1.50a software (National Institute of Health, USA; version 1.6.0_65).

Table 1.

Primary antibodies used in this study

Antibody raised against Purchased from Source Dilution
pFAK(Y397) BD Biosciences (Heidelberg, Germany) mouse 1:1000
FAK Cell Signaling (Danvers, USA) rabbit 1:1000
pSrc(Y418) BD Biosciences (Heidelberg, Germany) rabbit 1:1000
Src Biosource rabbit 1:1000
pAkt(S473) Cell Signaling (Danvers, USA) rabbit 1:1000
pAkt(T308) Cell Signaling (Danvers, USA) rabbit 1:1000
Akt Cell Signaling (Danvers, USA) rabbit 1:1000
pErk(T202/Y204) Cell Signaling (Danvers, USA) rabbit 1:1000
Erk Santa Cruz Biotechnology (Heidelberg, Germany) rabbit 1:750
pmTOR(S2448) Cell Signaling (Danvers, USA) rabbit 1:1000
mTOR Cell Signaling (Danvers, USA) rabbit 1:1000
pPDK1(S241) Cell Signaling (Danvers, USA) rabbit 1:1000
PDK1 Cell Signaling (Danvers, USA) rabbit 1:1000
pPTEN(S380/T382/T383) Cell Signaling (Danvers, USA) rabbit 1:1000
PTEN Cell Signaling (Danvers, USA) rabbit 1:1000
pRB(S807/S811) Cell Signaling (Danvers, USA) rabbit 1:1000
pRB(S795) Cell Signaling (Danvers, USA) rabbit 1:1000
actin Sigma-Aldrich, Steinheim, Germany mouse 1:4000
α-tubulin Abcam, Cambridge, UK mouse 1:5000
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NanoPro100™ assay

Capillary isoelectric focusing (cIEF) was performed as described previously [14]. Briefly, a nested G2 Premix, pH 5–8 (Protein Simple), pI (isoelectric point) standard ladder 3 (Protein Simple) and an additional 5.5 pI standard was prepared (master mix). According to a final protein concentration of 125 ng/capillary, Bicine/CHAPS as the sample diluent, DMSO inhibitor and the protein sample were added to the master mix. An anti-pAkt (S473) antibody (Cell Signaling, Danvers, USA) was used at a dilution of 1:25 and was detected using a HRP-coupled goat-anti-rabbit secondary antibody (Protein Simple) at a 1:100 dilution. The samples and antibodies were transferred to the assay plate and luminol/peroxide, washing buffer, catholyte and anolyte (all Protein Simple) were used according to the manufacturer’s protocol. The isoelectric focusing occurred for 40 min at 21 mW, followed by immobilization through UV exposure for 100 s. The primary antibody was incubated in the capillary for 4 h with two subsequent wash steps of 150 s each. The secondary antibody was incubated in the capillary for 1 h with two subsequent wash steps of 150 s each. Finally, the luminol/peroxide reagent was passed through the capillaries and chemiluminescence was detected. Peak integration and pI marker calibration were performed using Compass™ software as previously described [14, 50]. The cIEF graph shows a representative profile of the experimental condition.

Immunofluorescence microscopy

MCF-7 cells were seeded on cover slips (Roth, Karlsruhe, Germany) and grown to 60–70% confluence. Next, the cells were stimulated with 100 ng ml−1 IGF-1, with IGF-1 in the presence of Ino-C2-PAF or left untreated for 24 h. Subsequently, the cells were fixed with 4% paraformaldehyde in PBS for 20 min, blocked and permeabilized using 2% BSA, 1% goat serum, 1% sodium azide and 0.2% Triton X-100 in PBS for 1 h. MCF-7 cells were incubated with an anti-pAkt (S473) antibody (Cell Signaling, Danvers, USA) in blocking solution at 4 °C overnight. After extensive washing, an appropriate secondary goat anti-rabbit, Alexa Fluor 488-conjugated antibody (Invitrogen, Carlsbad, USA) was added for 1 h at room temperature. Cell nuclei were stained with Hoechst 33,342 dye (Sigma-Aldrich, Steinheim, Germany). After washing, the cover slips were mounted using ProLong Gold antifade reagent (Life Technologies, Eugene, USA) and analyzed using an Axiovert 200 microscope. Images were captured using an Axiocam at a magnification of 40× and processed using Axiovision software (release 4.8.2 SP3, Zeiss, Jena, Germany). For comparison purposes, images were acquired using identical settings of exposure and processing.

Hoechst staining was additionally used for the evaluation of cell proliferation. For this purpose, 10 randomly chosen images of IGF-1-treated (100 ng ml−1), IGF-1/Ino-C2-PAF-treated and untreated control cells, respectively, were analyzed using an Axiocam at a magnification of 25× for the presence of mitoses. The number of mitotic cells was set in relation to the total cell count and was expressed as % mitotic cells.

Statistical analyses

In each case, three independent experiments were performed and analyzed (unless stated otherwise). The arithmetic mean and standard deviation (SD) of the mean were calculated. P-values were determined using the two-tailed Student’s t-test. Indicators of significance correspond to p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***).

Results

Ino-C2-PAF inhibits the proliferation of MCF-7 and MDA-MB-231 cells at non-toxic concentrations

In previous studies, the impact of Ino-C2-PAF on cell growth was determined using the immortalized human keratinocyte-derived cell line HaCaT [8], the squamous carcinoma-derived cell line SCC-25 [7] and two lymphoma-derived cell lines [6]. To characterize the impact of Ino-C2-PAF on the breast cancer-derived cell lines MDA-MB-231 and MCF-7, we set out to determine their growth and viability in the presence or absence of Ino-C2-PAF. By monitoring lactate dehydrogenase (LDH) release in Ino-C2-PAF-treated and untreated cells, we found that Ino-C2-PAF was non-toxic or only slightly toxic in the range from 0.6 to 5 μM (with an proportion of dead cells clearly below the LC50 value) after an exposure time of 48 h (Fig. 1). At lower concentrations, MCF-7 cells were more sensitive towards Ino-C2-PAF than MDA-MB-231 cells. For MDA-MB-231 and for MCF-7 cells LC50/48h values of 7.4 μM and 9.3 μM, respectively, were observed. The lower LC50/48h value of MDA-MB-231 cells was found to be due to a rapid increase in cytotoxicity between 5 and 10 μM Ino-C2-PAF. The growth inhibitory properties of Ino-C2-PAF were also determined for both cell lines through the detection of BrdU incorporation after treatment for 48 h. We found that Ino-C2-PAF inhibited the growth of MDA-MB-231 cells in a concentration-dependent manner: 2.5 μM Ino-C2-PAF reduced their proliferation by approximately 38% and 5 μM Ino-C2-PAF reduced their proliferation by approximately 63% (Fig. 2a), Similarly, we found that the proliferation of MCF-7 cells was reduced by 2.5 μM Ino-C2-PAF to about 38% and by 5 μM Ino-C2-PAF to about 69% (Fig. 2b). For both cell lines, an IC50 of 3.5 μM was observed after 48 h incubation. To determine the effect of Ino-C2-PAF on anchorage-independent growth, the respective cells were subjected to a soft agar colony formation assay. As a negative control, untreated cells were used. Due to differences in growth behavior, the colony sizes of the MCF-7 and MDA-MB-231 cells were evaluated after 6 and 15 days, respectively. We found that 2.5 and 5 μM Ino-C2-PAF significantly inhibited the formation of colonies in soft agar in both cell lines compared to their untreated counterparts (Fig. 2b).

Fig. 1.

Fig. 1

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Ino-C2-PAF-mediated cytotoxicity on breast cancer-derived cells. The cytotoxicity of Ino-C2-PAF on MDA-MB-231 and MCF-7 cells was monitored by LDH release. Cells lysed with 2% Triton X-100 served as a positive control and were set at 100% cytotoxicity. Untreated cells were set at 0% cytotoxicity. Tests were performed in quadruplicate and were repeated at least three times. Error bars represent SD. ** indicates a highly significant difference (p < 0.01) compared to control cells

Fig. 2.

Fig. 2

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Effect of Ino-C2-PAF on breast cancer cell growth. a, b The proliferation rates of MDA-MB-231 and MCF-7 cells were determined by BrdU incorporation after 48 h at the indicated concentrations. Untreated cells were set at 100%. c Effects of Ino-C2-PAF on anchorage-independent growth of MDA-MB-231 and MCF-7 cells using a soft agar colony forming assay. The colony sizes were determined in the presence of 2.5 and 5 μM Ino-C2-PAF or in the absence of Ino-C2-PAF. All of the experiments were performed in quadruplicate and repeated three times. d Effects of Ino-C2-PAF on the motility of MDA-MB-231 and MCF-7 cells. Cells were allowed to migrate towards a collagen I gradient in serum-free medium in the presence or absence of 2.5 μM Ino-C2-PAF for 22 h. Three independent experiments were performed. Error bars represent SD. * a significant difference of p < 0.05, ** a significant difference of p < 0.01 and *** a significant difference of p < 0.001 compared to the control cells

Ino-C2-PAF attenuates the motility of MDA-MB-231 and MCF-7 cells

Most deaths caused by cancer are not due to the occurrence of primary tumors but to tumor invasion and metastasis, when cancer cells alter their migratory capacity and actively invade adjacent tissues and the vasculature. In vitro, cell motility can be studied using haptotactic migration of cells towards a matrix protein gradient. To assess whether the motility of MDA-MB-231 and MCF-7 cells is influenced by Ino-C2-PAF, the respective cells were treated with 2.5 μM Ino-C2-PAF, or were left untreated, and subjected to migration assays. The concentration of 2.5 μM was chosen since we found that it is non-toxic and that the invasion of cells was substantially inhibited by this concentration in preliminary experiments (Fig. S1). After detachment from the plate, the cells were allowed to migrate towards a collagen I gradient for 22 h at 37 °C in the presence or absence of Ino-C2-PAF. By doing so, we found that MDA-MB-231 cells exhibited a higher migration rate than MCF-7 cells (Fig. 2d; black bars). We also found that in the presence of Ino-C2-PAF the migration of both MDA-MB-231 and MCF-7 cells was clearly inhibited, i.e., from an average cell count of 129 to 61 and of 70 to 29, respectively.

Phosphorylation of Akt, but not of Erk1/2, FAK and Src, is inhibited by Ino-C2-PAF in MCF-7 and MDA-MB-231 cells

Several signaling molecules and pathways are known to play pivotal roles in regulating the growth and motility of breast cancer cells. These include the cytosolic tyrosine kinases FAK [15] and Src [16], the PI3K/Akt pathway [17, 18] and the Ras/Raf/MEK/ERK pathway [19]. Therefore, we set out to investigate whether Ino-C2-PAF has an impact on the phosphorylation status of key components within these pathways using Western blot analysis with phosphorylation site-specific antibodies in cell lysates of MCF-7 and MDA-MB-231 cells that were treated with 2.5 μM Ino-C2-PAF for 24 h or were left untreated. We found that, while phosphorylation of the MAP kinases Erk1/2, FAK and Src remained unaffected, Ino-C2-PAF inhibited the phosphorylation of Akt at serine residue 473 in both cell lines (Fig. 3a). We also analyzed HaCaT cells to evaluate whether this inhibitory effect on Akt phosphorylation could also be observed in other cells and, indeed, found that Ino-C2-PAF also inhibited Akt phosphorylation in these cells (Fig. 3b). Since the basic Akt phosphorylation levels in untreated cells could only be observed after longer exposure times of the Western blots, we addressed the question whether Ino-C2-PAF can also inhibit Akt phosphorylation if induced by a known activator. For this purpose, MCF-7 and MDA-MB-231 cells were treated with the growth factor IGF-1, which is an important modulator of cell growth, differentiation and invasion [18, 20] in the presence or absence of Ino-C2-PAF for 24 h or 48 h. We found that IGF-1 led to a substantial increase in Akt phosphorylation at serine residue 473. In the presence of Ino-C2-PAF this increase in Akt phosphorylation was robustly antagonized to basal levels. Again, we found that the phosphorylation of Erk1/2 remained unaffected (Fig. 3c). Densitometric quantification of pAkt and Akt signals and normalization of pAkt to the content of total Akt revealed that Ino-C2-PAF substantially inhibited the phosphorylation of Akt after IGF-1 treatment of both cell lines after 24 h and 48 h (Fig. 3d). We investigated this Ino-C2-PAF-induced inhibition of AktS473 phosphorylation in more detail in MCF-7 cells by indirect immunofluorescence analysis using an anti-pAktS473-specific antibody. Again, we found that IGF-1 substantially increased Akt phosphorylation at serine 473, while concomitant treatment with Ino-C2-PAF led to a clear reduction in phosphorylation to a basic level (Fig. 3e).

Fig. 3.

Fig. 3

Fig. 3

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Ino-C2-PAF attenuates the phosphorylation of Akt, but does not affect the phosphorylation of Erk1/2, FAK and Src. Total lysates of MCF-7, MDA-MB-231 (a) and HaCaT (b) cells that were treated with 2.5 μM Ino-C2-PAF or were left untreated for 24 h were immunoblotted using phosphorylation site-specific antibodies directed against pAktS473, pErkT202/Y204, pFAKY397 and pSrcY418 and the respective secondary antibodies. Antibodies directed against the unphosphorylated form of the respective kinases were used as loading controls. One representative of three independent experiments is shown. c Effect of Ino-C2-PAF on the phosphorylation of Akt in IGF-1-treated cells after 24 and 48 h. All experiments were repeated three times. Tubulin was used as a loading control. d Quantification of the signal intensities of pAktS473 and pErkT202/Y204 on the Western blots using Image J software. The data represent the mean of three independent experiments. * a significant difference of p < 0.05 and ** a significant difference of p < 0.01 compared to control cells. e Cells were kept under the same conditions as described in (c) and analyzed by indirect immunofluorescence for pAktS473. Out of three independent experiments, one example is shown. f Effect of Ino-C2-PAF on the phosphorylation of all pAktS473 positive forms expressed in MCF-7 cells. MCF-7 cell lysates of untreated cells or cells that were incubated with IGF-1 or IGF-1 plus Ino-C2-PAF for 24 h were analyzed by cIEF using an anti-pAktS473 antibody. The same cell lysates were analyzed in parallel by Western blotting for total Akt and (control) actin expression. In parallel, phosphorylation of pAktS473 is shown by Western blotting. The experiment was performed on two independent biological replicates with two technical replicates, each of which were considered separately in the evaluation. Error bars represent SD. ***p < 0.001. g Representative isoelectric focusing profiles with the isoelectric point on the X-axis and the Y-axis showing relative luminescence units. RLU = relative light units

We additionally performed a nanocapillary immunoassay, which is based on isoelectric focusing (cIEF), to separate proteins according to their net charge. By doing so, different phosphoforms of a protein can be separated and detected using specific antibodies. MCF-7 cells were left untreated or were stimulated with IGF-1 in the presence or absence of Ino-C2-PAF. Subsequent integration of the cIEF peak profiles revealed that IGF-1 increased the total phosphorylation at serine residue 473 by a factor of 10, while Ino-C2-PAF reduced the phosphorylation at this position to control levels (Fig. 3f). Detailed analysis of the cIEF peak profile showed that the increase in phosphorylation at S473 caused by IGF-1 particularly regarded Akt phosphoforms with isoelectric points (pIs) of 5.06, 5.14, 5.31, 5.42 and 5.53. We also found that treatment with Ino-C2-PAF reduced the phosphorylation of all of these phosphoforms to control levels (Fig. 3g). As mentioned earlier, the basic level of AktS473 phosphorylation is very low in MCF-7 cells (Fig. S2a). Therefore, the peak profile of Ino-C2-PAF-treated cells was only marginally decreased compared to the profile of control cells (Fig. S2b).

Ino-C2-PAF inhibits phosphorylation downstream of Akt while phosphorylation upstream of Akt remains unaffected

To be fully activated and stabilized, Akt requires phosphorylation at S473 and also at T308 [21]. To investigate whether Ino-C2-PAF has also an impact on the phosphorylation status at T308, MCF-7 cells were treated with IGF-1, in the presence or absence of Ino-C2-PAF, for 24 h and subjected to Western blot analysis. In contrast to Akt phosphorylation at S473, we observed sustained T308-Akt phosphorylation that was neither affected by the addition of IGF-1 nor by treatment of the cells with Ino-C2-PAF (Fig. 4a). Since we found that the phosphorylation of Akt was selectively inhibited by Ino-C2-PAF (see above), we next set out to investigate whether Ino-C2-PAF may affect signaling molecules upstream of Akt, i.e., PDK-1 and PTEN, using Western blot analysis. Under the conditions indicated, we found that Ino-C2-PAF did not affect the phosphorylation of both proteins (Fig. 4b). To additionally assess whether Ino-C2-PAF affects Akt-mediated downstream signaling in the presence or absence of IGF-1, we investigated the expression and phosphorylation of mTOR and pRb. We found that both signaling molecules exhibited increased phosphorylation levels in response to IGF-1 and strong decreases in phosphorylation levels in the presence of Ino-C2-PAF in unstimulated and IGF-1-stimulated MCF-7 cells (Fig. 4c).

Fig. 4.

Fig. 4

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Ino-C2-PAF attenuates the phosphorylation of Akt downstream effectors mTOR and pRB, but does not affect the phosphorylation of AktT308 or signaling molecules of the PI3K pathway that act upstream of Akt. Total cell lysates of MCF-7cells that were treated for 24 h with 2.5 μM Ino-C2-PAF, or were left untreated and IGF-1-treated in the presence or absence of Ino-C2-PAF were immunoblotted with phosphorylation site-specific antibodies directed against (a) pAktT308, (b) pPDK-1S241, pPTENS380/T382/T383 and (c) pmTORS2448, pRBS807/S811, pRBS795 and the respective secondary antibodies. Antibodies directed against the unphosphorylated forms of the respective kinases as well as actin were used as loading controls. One representative of three independent experiments is shown

Ino-C2-PAF distinctly inhibits IGF-1-induced proliferation in MCF-7 cells

It has long been recognized that high levels of IGF-1 in a tumor microenvironment (TME) may promote growth. Therefore, we set out to investigate whether the growth of MCF-7 cells induced by IGF-1 can be inhibited by Ino-C2-PAF. For this purpose, cells that reached 70% confluence were treated with IGF-1 alone or IGF-1 plus Ino-C2-PAF, or were left untreated for 24 h. Subsequently the cells were fixed, permeabilized and incubated with Hoechst to stain the nuclei. Next, the nuclei were analyzed for visible signs of mitosis. These mitotic cells (Fig. 5a; marked with an arrow) were defined as proliferating cells. Through the analysis of approximately 6000 cells under each experimental condition we found that the relative amount of proliferating cells in the control cells was 3.75%, while IGF-1-treatment increased the amount of proliferating cells to 5.52%. The concomitant treatment with Ino-C2-PAF decreased the number of mitotic cells to 2.61% (Fig. 5a). We also investigated whether IGF-1 has an impact on haptotactic cell migration in MCF-7 cells. Cells that were subjected to the migration assay were treated with IGF-1, or IGF-1 plus Ino-C2-PAF or were left untreated for 24 h, after which the cells were allowed to migrate towards a collagen I gradient. We found that under these conditions IGF-1 had no impact on the migration towards collagen I in MCF-7 cells (Fig. 5b). We also found that the decrease in migration in the presence of Ino-C2-PAF was similar to that observed in unstimulated cells (Fig. 2d) and that this migration, thus, appears to be independent of the IGF-1 pathway.

Fig. 5.

Fig. 5

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Ino-C2-PAF inhibits IGF-1-induced proliferation and, to a lesser extent, IGF-1-induced migration. a, b 6389 nuclei of unstimulated MCF-7 cells, 5535 nuclei of IGF-1-treated MCF-7 cells and 5688 nuclei of IGF-1/Ino-C2-PAF-treated MCF-7 cells were analyzed for mitotic features. Cells in prophase, metaphase, anaphase and telophase were regarded as mitotic, whereas cells in cytokinesis were excluded. Four independent experiments were performed. c MCF-7 cells that were treated with IGF-1 alone, IGF-1 and 5 μM Ino-C2-PAF, or that were left untreated were allowed to migrate towards a collagen I gradient for 24 h. Three independent experiments were performed. Error bars represent SD. * p < 0.05 and **p < 0.01 compared to controls

Discussion

Due to their molecular complexity and acquisition of resistance to existing drugs, novel strategies for the treatment of breast cancer, particularly advanced and metastatic breast cancer, are required. Inositol-C2-platelet activating factor (Ino-C2-PAF) belongs to a group of synthetic APLs, amphiphilic glycerophospholipids, that are structurally derived from PAF. In contrast to commonly used cytostatic compounds such as cisplatin or vincristine, APLs do not interfere with DNA or the mitotic spindle apparatus of the cell, but due to their lipophilic character, ALPs are incorporated into cell membranes and, as such, they represent a unique therapeutic entity [22–24]. It has been demonstrated that the prototype of APL, edelfosine, interferes with the proliferation of tumor cells, such as multiple myeloma and leukemia cells, by targeting lipid rafts and a subsequent induction of apoptosis [25, 26]. In addition, it has been reported that APLs in combination with radiotherapy represent a promising strategy to improve the clinical outcome of cancer [27, 28]. It has been found that Ino-C2-PAF may also inhibit proliferation and migration in immortalized keratinocytes and squamous cell carcinoma cells in a more efficient manner than other APLs such as edelfosine [7, 8].

In the present work, we show that Ino-C2-PAF can inhibit the proliferation of both ER-positive and PR-positive MCF-7 cells and triple-negative MDA-MB-231 cells at an IC50 that is clearly below its toxic concentration. These data suggest that Ino-C2-PAF exhibits a similar or even stronger anti-proliferative effect than other APLs such as perifosine and miltefosine, even if these outcomes may be affected by different experimental conditions and detection methods used [29, 30]. Furthermore, it has been shown that the structurally related compounds edelfosine and erufosine can inhibit the growth of MCF-7 and MDA-MB-231 cells to a similar extent as Ino-C2-PAF [31, 32].

The progression of cancer, and particularly of those with high metastasis rates such as breast cancer, is not only governed by cell proliferation, but also by cell migration and invasion. Although the anti-proliferative impact of APLs has been widely studied, the effects of these compounds on cell migration and invasion is less well investigated, although recent data have pointed at an inhibitory role of APLs in cancer cell migration [33–35]. Here, we also observed an anti-migratory activity of Ino-C2-PAF on MDA-MB-231 and MCF-7 cells. In both cell lines, haptotactic migration towards a collagen I gradient was reduced to approximately 50%. Other reports on haptotactic migration towards a collagen I gradient indicated that Ino-C2-PAF and glucose-PAF (another member of the glycosidated phospholipid family) may markedly reduce the migration of keratinocytes [7, 36]. Similar results were obtained using in vitro wound-healing assays [7]. By contrast, the glycosidated phospholipids glucose-PC and glucosamine-PAF have been found to increase the migratory capacity of keratinocytes, whereas naturally occurring PAF did not affect migration in this cell system [36]. A negative effect on motility has also been reported for other APLs [37–39], whereas others reported an induction of invasion caused by edelfosine [40], especially in breast cancer cells [41, 42]. The edelfosine-induced invasion observed in these studies was accompanied by an increased activation of both Src and FAK [42]. Our data show independence of FAK and Src of the Ino-C2-PAF mode of action in breast cancer cells and, therefore, they are indicative of cell type-specific effects of this compound.

Ino-C2-PAF-mediated inhibition of Akt phosphorylation at Ser 473 was observed in all cell lines tested, including MCF-7, MDA-MB-231 and HaCaT, also upon IGF-1 activation, while phosphorylation of the MAP kinase Erk1/2 remained unaffected under the same conditions. Therefore, we conclude that the impact of Ino-C2-PAF on pAkt S473 may be more general and cell-type independent. We found that IGF-1 treatment increased phospho-Akt peaks from pI 5.06 to 5.53 in the cIEF, which were all decreased by Ino-C2-PAF. Deduced from recently obtained data, we suggest that the most acidic phospho peaks (pI 5.06–5.31) correspond primarily to Akt 1, while peaks with pI 5.42 and 5.53 correspond to mixed Akt isoforms, most likely Akt 1 and Akt 2 [14]. Therefore, we conclude that Ino-C2-PAF has no preference for pAktS473-specific phosphoforms or Akt isoforms. Targeting Akt activity implies that the PI3 kinase pathway, which initiates Akt phosphorylation, is selectively affected by Ino-C2-PAF. Among APLs, it has repeatedly been shown that perifosine can inhibit the PI3K signaling cascade, thereby exhibiting significant anti-proliferative activities in vitro and in vivo in several human tumor model systems [43, 44]. Phosphorylation of Akt has also been found to be affected by edelfosine and miltefosine in a dose-dependent manner in epithelial carcinoma-derived cell lines [45, 46]. Paradoxically, we found that only phosphorylation at S473 is inhibited by Ino-C2-PAF, whereas phosphorylation at T308 is not affected. Phosphorylation of T308 is due to a PI3K-dependent localization of PDK1 to the membrane. This indicates that PI3K is not directly targeted by Ino-C2-PAF. This notion hints at mTOR kinases, which may be targeted by Ino-C2-PAF in MCF-7 cells. mTOR exists in two complexes: mTORC1 and mTORC2. mTORC1 controls cell size and protein translation downstream of Akt, while mTORC2 has been shown to control the phosphorylation of Akt at S473. This mTORC2-dependent phosphorylation of Akt at S473 enhances the catalytic activity of Akt already phosphorylated at T308. Thus, mTOR complexes function both upstream and downstream of Akt [47]. Here we show that APLs, especially Ino-C2-PAF, can selectively inhibit the phosphorylation of Akt at S473, without affecting its phosphorylation at T308 in breast cancer-derived cells. This inhibition, in turn, results in inhibition of mTORC1, which normally is involved in cell cycle regulation since it activates the translation and formation of cyclin/cyclin-dependent kinase (cdk) complexes. These complexes phosphorylate the tumor suppressor protein pRb at Ser795 and S807/S811 [48], which leads to activation of the transcription factor E2F, and to entry into the S-phase. Consistent with our data, it was recently shown that a chemically similar compound, D-3-deoxydioctanoylphosphatidylinositol (D-3-deoxy-diC8PI) can decrease the levels of endogenous cyclin D1 and cyclin D3 in MCF-7 cells, inactivate pRB and reduce DNA synthesis [49]. We hypothesize that Ino-C2-PAF targets the Akt/motor/pRB axis, which results in a growth arrest in MCF-7 cells.

Due to the different mode of action of Ino-C2-PAF compared to other cytostatic drugs, this work highlights a novel concept for the treatment of ER-positive and -negative breast cancers. Whether Ino-C2-PAF is also efficacious in other breast cancers (e.g. HER2-positive) remains to be established in suitable preclinical models.

Electronic supplementary material

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Acknowledgements

We would like to thank Dr. Göran Landberg (Sahlgrenska Cancer Center, University of Gothenburg, Sweden) for providing the MDA-MB-231 and MCF-7 cell lines used in this study. This work was supported by the Berliner Krebsgesellschaft (grant to KD).

Authors‘ contributions

Participated in research design: Danker, Hildmann, Klein, Eickholt and Lucka. Conducted experiments: Pelz, Häckel, Semini. Schrötter, Stricker, Bintig and Mrawietz. Synthesis of Ino-C2-PAF: Shmanai. Performed data analysis: Pelz, Häckel and Hildmann. Wrote the manuscript with input from all of the authors: Danker, Häckel and Pelz. Supervision the entire study: Danker.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interests.

Research involving human paricipants and/or animals

This article does not contain any studies with human participants or animals performed by any of the authors.

Informed consent

For this type of study informed consent is not required

Footnotes

Carsten Pelz and Sonja Häckel contributed equally to this work.

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