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Lipopeptide Biosurfactant Pseudofactin II Induced Apoptosis of Melanoma A 375 Cells by Specific Interaction with the Plasma Membrane

In the case of melanoma, advances in therapies are slow, which raises the need to evaluate new therapeutic strategies and natural products with potential cancer cell inhibiting effect. Pseudofactin II (PFII), a novel cyclic lipopeptide biosurfactant has been isolated from the Arctic strain of Pseudomonas fluorescens BD5. The aim of this study was to investigate the effect of PFII on A375 melanoma cells compared with the effect of PFII on Normal Human Dermis Fibroblast (NHDF) cells and elucidate the underlying mechanism of PFII cytotoxic activity. Melanoma A375 cells and NHDF cells were exposed to PFII or staurosporine and apoptotic death was assessed by monitoring caspase 3-like activity and DNA fragmentation. From time-dependent monitoring of lactate dehydrogenase (LDH) release, Ca2+ influx, and a correlation between Critical Micelle Concentration (CMC) we concluded that cell death is the consequence of plasma membrane permeabilisation by micelles. This finding suggests that pro-apoptotic mechanism of PFII is different from previously described cyclic lipopeptides. The mechanism of PFII specificity towards malignant cells remains to be discovered. The results of this study show that PFII could be a new promising anti-melanoma agent.


Introduction
Melanoma is the most aggressive form of skin cancer with a median survival time of 8-9 months and a 3-year survival rate of 10%-15%. In Europe, dacarbazine (DTIC) is the only approved drug for use as a systemic therapies for melanoma lesions [1][2][3][4]. Most of the cytotoxic anticancer drugs in current use have been shown to induce apoptosis in susceptible cells [5]. Apoptosis, which is a major pathway of programmed cell death, plays essential roles in the maintenance of homeostasis and tissue development in organisms. The major apoptotic pathways have been identified as the death receptor-mediated pathway, the mitochondrial apoptotic pathway, and the endoplasmic reticulum pathway [6][7][8][9].
Biosurfactants are biological surface-active compounds with both hydrophilic and hydrophobic moieties produced by diverse microorganisms. Several bioactive natural surfactants, e.g., lipopeptide and glycolipid have been found to possess antibacterial, antifungal, anti-viral, hemolytic and ionophoric properties [10][11][12]. Some of these molecules have been shown to induce apoptosis in tumor cells. For example, a lipopeptide produced by Bacillus subtilis has anti-tumor activity on LoVo cells [13] and a cyclic lipopeptide from Bacillus natto T-2 induces apoptosis in human leukemia K562 cells [14] by an increase in [Ca 2+ ] and Extracellular Signal-regulated Kinases (ERK) phosphorylation [15]. Glycolipids from Candida antarctica and their analogs have been implicated in growth arrest, apoptosis, and differentiation of mouse malignant melanoma and human HL60 cells [16,17].
We have previously purified and identified two new cyclic lipopeptides -pseudofactin I and pseudofactin II from Pseudomonas fluorescens BD5 [18,19]. Both compounds are cyclic lipopeptides (Gly-Ser-Thr-Leu-Leu-Ser-Leu-Leu/Val) with a palmitic acid connected to the terminal amino group of the octapeptide. The C-terminal carboxylic group of the last amino acid (Val or Leu) forms a lactone bond with the hydroxyl of Thr3. Previously we reported that pseudofactin II (PFII) lowered the adhesion and partially dislodged biofilm of five bacterial species and Candida albicans [20]. The aim of this study was to examine whether and how PFII affects melanoma cells. We have found that PFII induces apoptosis in melanoma (A375) cell line and that its biosurfactant activity is most effective above critical micelle concentration (130-140 mM).

Cell Proliferation
The proliferation rate of melanoma A375 cells and normal human dermal fibroblast (NHDF) cells grown in the presence of PFII was measured by MTT assay. A dose-dependent decrease in melanoma cell viability was observed with increasing (7 mM to 260 mM) concentrations of PFII ( Figure 1A). The highest concentrations of PFII (180 mM and 260 mM) caused a nearly complete eradication of melanoma cells. In contrast, the viability and proliferation of NHDF and NHEK cells were less affected by PFII. NHDF and NHEK cells exposed to the highest PFII concentration (260 mM) and/or longer incubation times resulted in an approximate 50% decrease in proliferation rate ( Figure 1B, C).

Nuclei Fragmentation
To evaluate the potential pro-apoptotic activity of PFII on melanoma A375 cells, the integrity of PFII-treated melanoma cell nuclei was examined using fluorescence microscopy after Hoechst 33342 staining (Figure 2A). The results were compared with those observed in the presence of staurosporine (STS), a known apoptotic agent. Melanoma cells grown in the presence of PFII or STS exhibited changes characteristic for apoptosis (Figure 2A, B, C), i.e., fragmented and 'moon-shaped' nuclei ( Figure 2A, white arrows). We did not observe any changes in the nuclear integrity of NHDF cells cultured in the presence of the same concentrations of PFII (data not shown).

Actin Condensation and Caspase-3 Activation
Melanoma A375 cells cultured in the presence of PFII reorganized their actin cytoskeleton. The lowest concentration of PFII (65 mM) employed induced a sub-membrane condensation of filamentous actin (F-actin). At higher concentrations of PFII (130-260 mM) the cells became rounded, exhibited F-actin disorganization, strong sub-membrane condensation, and bleb formation. The actin cytoskeleton of melanoma cells cultured in the presence of STS was almost completely disorganized and actin ''aggregates'' were visible, even at the lowest tested concentration (65 mM) after 24 hours of incubation ( Figure 3F-J). The disruption of nuclear integrity, cyto-morphological changes, and actin cytoskeletal disorganization in melanoma cells grown in the presence of PFII were accompanied by caspase-3 activation similar to that observed in melanoma cells cultured in medium supplemented with STS ( Figure 3A-E). The normal human dermal fibroblast, which were used as a control, did not respond to the PFII treatment with induction of any of the applied apoptotic markers. When comparing images G, H and J, K of Figure 4, we did not observe significant alterations in the distribution of F-actin and increase in caspase-3 activity in NHDF cells grown in medium with PFII. In contrast, staurosporine induced the apoptotic changes in NHDF cells (Figure 4 I, L).

DNA Fragmentation
The effect of PFII on DNA fragmentation in melanoma cells is in Figure 2B. A ''ladder'' pattern representing fragmentation of melanoma cell DNA into oligonucleosome length fragments was observed after 24 h of PFII treatment. We also observed internucleosomal chromatin cleavage in melanoma cells treated with 1 mM staurosporine for 2 h ( Figure 2C). Treatment of melanoma cells with PFII or staurosporine resulted in similar DNA fragmentation patterns.

Calcium Influx
Fluo-3-AM fluorescent probe labeled melanoma cells treated with PFII, as well as apoptotic cells in the presence of STS, exhibited significant Ca 2+ influx ( Figure 5). NHDF cells, which were used as a control, did not respond to PFII treatment with induction of the Fluo-3/AM ( Figure 4A, B). In contrast, staurosporine was able to induce apoptotic changes in NHDF cells following two hours of incubation ( Figure 4C).

Annexin V Staining of Apopotic Melanoma A375 Cells
Labeling with fluorescent annexin V was used to detect the presence of phosphatidylserine in the external layer of the plasma membrane. After treatment with PSII at concentrations (65, 130 and 260 mM) for 24 h a large number melanoma A375 cells were positively stained by annexin V-fluorescein ( Figure 6B-D), whereas we did not observe this effect in NHDF cells  ( Figure 4E). Staurosporine at a concentration of 1 mM caused annexin V labeling in a manner similar to that observed with PFII ( Figure 6E).

Membrane Integrity
Cell death may be associated with impairment of membrane integrity resulting from interaction with biosurfactants (monomeric or micelles). This was examined by monitored the release of lactate dehydrogenase (LDH) into the extracellular milieu following exposure to increasing concentrations of PFII for 4 or 24 h (Figure 7). PFII supplementation above a concentration 130 mM increased cell death in melanoma cells. The cytotoxic effect of PFII at concentration of 130 mM intensified with exposure time for melanoma cells, but not for NHDF cells. At a PFII concentration of 260 mM nearly all melanoma cell were killed within 4 h, while 50% of NHDF cells were still viable after 24 h.

CMC Evaluation in Culture Medium
To explain the increase of pro-apoptotic activity at concentrations above 130 mM, PFII surface tension was measured in DMEM and a-MEM medium by the du Nouy's ring method [19]. The critical micelle concentration (CMC) was estimated to be in the range 130-140 mM. This suggests that the mechanisms of apoptosis induction by PFII in melanoma cells may be strongly dependent on micelles formation.

Measurements of Mean Diameter of PFII Micelles
In this study biosurfactant-PFII was applied at concentrations higher than the critical micellar concentrations so as to relate their respective micellar properties to their potential proapoptotic effects. The physicochemical properties of the polymeric micelles were examined in terms of particle size and polydispersity index (PDI) (data not shown). The mean hydrodynamic diameter of PFII micelles in water was ranged from 40.2 nm to 60.3 nm, while PDI values ranged from 0.137 to 1.0.

Discussion
The lipopeptide biosurfactant pseudofactin II induces apoptosis of melanoma A375 cells, while normal human dermal fibroblast are much less affected under the same experimental conditions. The mechanism of apoptosis induction may be based on the membrane permeabilisation [21] resulting from the interaction of PFII micelles with the plasma membrane, as evidenced by the release of LDH into the culture medium ( Figure 7). Thus, the mode of action of PFII is different from pro-apoptotic activity of the previously described cyclic lipopeptide (CLP) [15] which did not affected membrane integrity. Impairment of membrane integrity may lead to the observed Ca 2+ influx, which triggers further signaling cascades, like measured caspase-3 activation, that finally lead to apoptosis. This hypothesis of the mechanism of melanoma cells death by PFII is further supported by the non-linear increase in cytotoxic activity of PFII above CMC. Usually the CMC of surfactants is measured in water, as it was previously determined for PFII [19]. However, due to interactions PFII with cell culture medium components, CMC measured in our experimental conditions was about 2-fold higher (130-140 mM) than previously measured in water. This concentration correlated very well with the increase of PFII pro-apoptotic activity (Figure 1). While the suggested mechanism of action seems very likely, PFII specificity against melanoma cells remains to be elucidated. In vitro studies using trehalose lipid biosurfactant suggests that it acts as a weak detergent which may prefer membrane incorporation over micellization. Trehalose lipid biosurfactant also exerts hemolytic activity [22], while this was not the case for PFII (results not shown). The specific activity of PFII toward melanoma cells could result from a variation in plasma membrane composition, for example, lower sterol content. The increasing melanoma incidence among the general population (associated with global decreases in stratospheric ozone and increased recreational exposure) is alarming [23] and is most often due to late diagnosis. The prognosis for individuals with this disease for curing is relatively poor. Thus, PFII could be considered as important role in promoting apoptosis at the very early stages of the melanoma formation or in prevention of its expansion. Thus, the PFII could be further analyzed on human skin for the ability to induce irritant or allergic contact dermatitis.

Cell Treatment
Melanoma A375 cells, NHDF cells and NHEK cells were treated for 6 (data not shown), 24

Visualization of Actin Microfilaments, Caspase-3 Activity, and Nuclear Morphology
Melanoma A375 and NHDF cells grown on sterile glass coverslips were fixed with 4% paraformaldehyde (PFA) in PBS at 4uC for 20 min, rinsed (3X) with PBS, and then permeabilized with a 0.1% Triton X-100 in PBS for 20 min at room temperature (RT). The cells were rinsed with PBS and incubated in 1% bovine serum albumin in PBS for 1 h at RT. Active caspase-3 was labelled with monoclonal anti-caspase-3 antibody (clone 269518; R&D Systems) at a concentration of 1 mg/ml, followed by treatment with FITC (fluorescein isothiocyanate)-conjugated anti-mouse secondary antibody (Jackson Im-munoResearch) diluted in PBS (1:200).
Apoptotic nuclear morphology of fixed cells was assessed using Hoechst 33342 (Molecular Probes, USA), which was applied at a concentration of 5 mg/ml for 10 min incubation.
The cells (coverslips) were washed with PBS, mounted with fluorescence stabilizing medium (Dako, Glostrup, Denmark). Depending on the above experiment, observations were made with an Olympus IX70 fluorescence microscope or an Olympus FV 500 laser scanning confocal microscope (LSCM).

DNA Fragmentation
A375 melanoma cells (3610 5 cells) were seeded in 6-well microtiter plates and cultured in DMEM medium supplemented with vehicle (DMSO 1%) or DMSO containing PFII (65, 130, 260 mM) for 24 h at 37uC. Staurosporine (STS), a classic inducer of apoptosis, was used as a positive control. Cells were treated in medium containing 1 mM STS for 2 h at 37uC. After treatment with PFII or STS, the cells were scraped from the dishes using a rubber policeman and centrifuged at 90006g for 5 min at 4uC. The pellet was washed (2X) with ice-cold PBS and the DNA extracted using a NucleoSpin Tissue kit (Macherey-Nagel, Düren, Germany) according to the manufacturer's instructions. The DNA fragments obtained were electrophoretically separated using a 1.5% agarose gel. Gels were stained with ethidium bromide and photographed under UV transillumination.

Lactate Dehydrogenase (LDH) Assay
The release of LDH from damaged cells was measured with CytoTox-ONE (Promega, MA, USA) homogeneous membrane integrity assay. Melanoma A375 and NHDF cells were seeded onto 96-well plates at a density of 1610 4 and 3610 3 cells per well, respectively. Cells were maintained in humidified incubator at 37uC and 5% CO 2 . The cells were incubated for 2 and 24 h with increasing concentrations (65, 130, 260 mM) of PFII added to the cell culture medium. Maximum LDH release was determined by adding 2 ml of the CytoTox-ONE lysis solution to control wells for 10 min. The assay was performed in 96-well plates by adding 100 ml of the sample supernatant and 100 ml of CytoTox-ONE reagent, after which the plate was shaken for 10 s. After 10 min of incubation, 50 ml CytoTox-ONE stop solution was added and the plate was again shaken for 10 s. The fluorescent signal was measured with a Cary Eclipse Spectrofluorimeter (Varian, Cary, NC) with l EX 560 nm, l EM 590. LDH-release was calculated as percentage of LDH released in the culture media of total LDH (media and lysates).

Measurement of Micelle Size
The mean particle size and polydispersity index (PDI) of PFII diluted in Milli-Q water were determined using a Zetasizer Nano-ZS (Malvern Instruments Ltd., Malvern, UK) and PCS software. The analysis of particle size and PDI, determined by photon correlation spectroscopy, was performed using the volume distribution algorithm. The polydispersity index qualifies the particle size distribution, which here ranged from 0 for monodispersed to 1.0 for entirely heterodispersed emulsions. In order to obtain the optimum light scattering intensity for the size analysis, approximately 100 ml of the PFII was added to 900 ml of Milli-Q water. All the measurements were carried out at 25uC.

Statistical Analysis
All data are presented as mean 6 standard deviation (SD). Statistical significance was determined using Student's t test. The significance level was set at P,0.05.