Inhibitory Effects of Hybrid Liposomes on the Growth of Non-small Cell Lung Carcinoma Cells and Anti-invasive Activity by Ceramide Generation without any Drugs

Ceramide generation and invasive inhibition by hybrid liposomes (HL) composed of 90 mol% L-α-dimyristoylphosphatidylcholine (DMPC) and 10 mol% polyoxyethylene (23) dodecyl ether (C12(EO)23) were examined for human lung cancer (A549) cells in vitro. Ceramide generation for A549 cells treated with HL was observed through the activation of neutral sphingomyelinase. These was a reduction in sphingomyelin in A549 cells after treatment with HL. The anti-invasive effects of HL for A549 cells were obtained through the inhibition of MT1MMP/MMP14. Inhibitory effects of HL on the migration of A549 cells was obtained using scratch assay.


Introduction
Non-small cell lung carcinoma (NSCLC) is the most common form of lung cancer, which accounts for up to 85% of deaths [1,2]. Metastasis in NSCLC is the major cause of mortality among patients [3,4]. Lymph node metastasis and invasion to neighbouring organs are as the most important indicators of poor prognosis [4,5]. In the recent years, several potential oncogenic drivers have been identified in patients with NSCLC, including epidermal growth factor receptor (EGFR), B-Raf (BRAF), Kirsten rat sarcoma 2 viral oncogene homolog (KRAS), mesenchymal epithelial transition factor (MET), human epidermal growth factor receptor 2 (HER2), and anaplastic lymphoma kinase gene (ALK) [6][7][8][9]. The inhibitors for these genes such as the gefitinib and crizotinib are used in the treatment for the patients with NSCLC [8,10,11]. However, these drugs for NSCLC have adverse events [10,11]. Therefore, novel treatment strategies directed against NSCLC are needed.
Furthermore, HL induced cell cycle arrest at G 0 /G 1 phase in NSCLC cells through the inhibition of Akt signaling [26]. In addition, it has been reported that HL treatment altered the amounts of sphingomyelin and ceramide [27]. Ceramide is a sphingolipid-derived second messenger in cell the membrane in response to inflammation and stress [28]. It is related to cellular differentiation, proliferation and apoptosis pathways [28,29].

Dynamic light scattering measurements
The diameter of HL was measured using an electrophoretic light scattering spectrophotometer (ELS-Z0, Otsuka Electronics, Osaka, Japan). The diameter (dhy) was calculated using the Stokes-Einstein formula (Equation 1), where κ is the Boltzmann constant, T is the absolute temperature, η is the viscosity and D is the diffusion coefficient:

Ceramide assay by confocal laser microscope
Cells were seeded at a density of 4.0 × 10 3 cells pre well in 8-well chamber slides (Lab-Tek II, Thermo Fisher Scientific, MA, USA) and incubated for 24 hours. Then, HL was added (200 and 300 μM) and the plates were incubated for 3 hours. The cells were washed and directly fixed with 10% formaldehyde for 30 min in the wells. Cells were washed and permeabilized with ice-cold 100% methanol for 10 min at -20°C. Cells were blocked with 5% goat serum in PBS(-). After that, cells were washed twice and incubated with anti-ceramide mouse IgM monoclonal antibody (diluted 1:250, Enzo Life Sciences, NY, USA) at 4°C for 18 hours and Alexa Fluor 488 ® Goat Anti-Mouse IgG antibody (diluted 1:200, Invitrogen, CA, USA) in humidified box at room temperature for 1 hours. Finally, the cells were counterstained with TO-PRO-3 (Invitrogen, CA, USA). The stained cells were observed using a confocal laser microscope (TCS-SP; Leica Microsystems, Wetzlar Germany) with a 488 nm Ar laser. The Alexa Fluor 488 ® fluorescein signals were detected at 515-565 nm and the TO-PRO-3 signals were detected at 649-740 nm.

Ceramide assay with sphingomyelinase by flow cytometry
Ceramide expression was detected by immunocytochemistry using a flow cytometer. Cells were seeded at a density of 1.0 × 10 5 cells per well in dishes and incubated for 24 hours. Cells were pretreated with GW4869 (neutral sphingomyelinase (nSMase) inhibitor, 3 μM, Merck Millipore, MA, USA) for 30 min [30,31]. Cells were treated with HL (300 and 500 μM) for 3 hours. Briefly, cells were washed with PBS (-), trypsinized, centrifuged and fixed with 10% formaldehyde for 30 min in the wells. Cells were washed and permeabilized with 100% methanol for 5 min on ice. After that, cells were washed twice and incubated with anti-ceramide mouse IgM monoclonal antibody (diluted 1:200) at room temperature for 30 min and Alexa Fluor 488 ® Goat Anti-Mouse IgG antibody (diluted 1:200) in humidified box at 4°C for 1 h. Cells were washed and measured by a flow cytometer.

Sphingomyelin assay
Sphingomyelin in cell were detected using sphingomyelin quantification colorimetric assay kit (Bio Vision, CA, USA) according to the manufacturer's directions. Cells were seeded at a density of 1.0 × 10 5 cells per well in dishes and incubated for 24 hours. Cells were treated with HL (300 μM) for 24 hours. Cells were washed with PBS(-), trypsinized and centrifuged. Then, collected cells were homogenized for three times using ultrasonic cell crushing apparatus (XL2020, Misonix ASTRASON, MISONIX INC., NY, USA). Homogenate was centrifuged and heated at 70°C. Membrane lipids were extracted from heated homogenate. After that, sphingomyelin in extract was stained with highly specific probe (OxiRed Probe, Bio Vision, CA, USA) and measured at absorbance (570 nm) in a microplate reader (Molecular Devices, CA, USA).

Invasion assay in vitro
In vitro cancer cell invasion was assessed using a Biocoat ™ Matrigel ™ invasion chamber (BD Biosciences, NJ, USA) with cell culture inserts containing an 8 μm pore size membrane with a thin layer of Matrigel basement membrane matrix. Briefly, 450 μl of A549 cells (4.0 × 10 4 cells/ml) were re-suspended in a serum-free medium containing either 5% glucose solution (control) or HL (100, 200, 300 μM) which was added to the cell culture insert of a Biocoat ™ Matrigel ™ invasion chamber. Then, 0.7 ml of medium supplemented with 10% FBS was added in the outer chamber as a chemoattractant. The cells were then incubated at 37°C in humidified 5% CO 2 conditions for 24 h. To quantitative tumor cell invasion, non-invading cells were removed from the upper surface of the membrane by scrubbing gently with a cotton-tipped swab. The cells on the lower surface of the membrane were fixed with ethanol and stained with crystal violet. The cells were photographed using a light microscope (ECLIPSE TS100, Nikon, Tokyo, Japan). Microscope fields of the lower surface of the membrane were counted for numbers of cells that had invaded through the Matrigel layer and the membrane. The results were expressed as numbers of invaded cells per microscope field.

Assessment of MT1-MMP (MMP-14) by flow cytometry
A549 cells were seeded at a density of 1.0 × 10 5 cells per well in dishes and incubated for 24 hours. Cells were treated with HL (300 and 500 μM) for 3 and 24 hours. Cells were washed with PBS (-), trypsinized, centrifuged at 1500 rpm × 5min and fixed with 10% formaldehyde for 30 min in the wells. Cells were permeabilized with cold methyl alcohol in PBS (-) for 30 min on ice. After that, cells were washed twice and incubated with anti-MMP14 rabbit monoclonal antibody (Abcam plc, Cambridge, UK) (5 μg/ml) at room temperature for 30 min and Alexa Fluor 488 ® Goat Anti-Mouse IgG (10 μg/ml) antibody in humidified box at 4°C for 1 h. Cells were washed and measured by a flow cytometer.

Scratch wound assay in vitro
A549 motility was assessed using a scratch wound assay. The cells were seeded into tissue culture dishes at a concentration of 2.5 × 10 5 cells/ml and cultured in medium containing 10% FBS to nearly confluent cell monolayers, which were then carefully wounded using a 200 μl sterile pipette tip, and any cellular debris was removed by washing with PBS(-). The wounded monolayers were then incubated in medium containing 10% FBS and treated with or without 100 μM HL. After 19 h, the cells were photographed under a light microscope (EVOS fl, Life Technologies, CA. USA). The migration areas were estimated using image analysis software ImageJ (Version 1.46r, National Institutes of Health, Bethesda, MD, USA).

Statistical analysis
Results are presented as mean ± S.D. Data were statistically analyzed using Student's t-test. A p value of less than 0.05 was considered to represent a statistically significant difference.

Physical properties of HL
We examined the morphology of HL composed of 90 mol% DMPC and 10 mol% C 12 (EO) 23 on the basis of dynamic light scattering measurements. The time course of the hydrodynamic diameter (dhy) change for HL was examined using an electrophoretic light scattering spectrophotometer. The mean dhy of HL stored at room temperature (25°C) was under 100 nm with a single and narrow distribution and remained stable for more than one month. In contrast, DMPC liposomes were unstable and precipitated after 14 days. HL were kept at room temperature due to the convenience of stocking for a long term period prior to clinical application. It is worthwhile to note that HL being under 100 nm in diameter could avoid the reticular endotherial system (RES) [32] and thus should be appropriate for intravenous administration in vivo and clinical applications.

Effects of HL on ceramide generation
We examined the effects of HL on ceramide generation in A549 cells on the basis of immunocytochemistry using a confocal laser microscope. The results are shown in Figure 1. Increase ceramide generation was observed in A549 cells after the treatment with HL in a dose-dependent manner. We have already reported that HL treatment altered the amounts of ceramide [27]. These results strongly suggest HL induced ceramide generation in A549 cells.

Effects of HL on sphingomyelinase in vitro
Ceramide can also be generated in the plasma membrane by the action of SMases [29]. We examined the effects of HL on the activating of neutral sphingomyelinase (N-SMase) using GW4869 nSMase inhibitor by flow cytometric analysis in vitro. The results are shown in Figure 2. Inhibition of ceramide generation in A549 cells treated with HL with GW4869 N-SMase inhibitor (p<0.05) was obtained, although an increase of the ceramide in A549 cells after treatment with HL was observed in a dose-dependent manner. These results indicate that ceramide generation by HL in A549 cells was involved in the activation of N-SMase.

Effects of HL on sphingomyelin
Ceramide are synthesized from sphingomyelin in the plasma membrane by activation of N-SMases [29]. We examined the amount of sphingomyelin in A549 cells using a sphingomyelin quantification colorimetric assay kit in vitro. The results are shown in Figure 3. The amount of sphingomyelin in A549 cells after the treatment with HL decreased (p<0.05). This result suggests that HL could synthesize the ceramide from sphingomyelin by activation of N-SMases in vitro. Furthermore, we are now investigating mechanism of ceramide generation by HL in detail.

Inhibitory effects of HL on invasion
Tumor cell invasion is an important early step in the process of tumor metastasis. In vitro tumor cell invasion was performed on the invasion assay using a Matrigel invasion chamber. The results are shown in Figure 4. The invasion rate of A549 cells treated with HL significantly decreased in a dose-dependent manner (Figures 4A and  4B). Interestingly, the anti-invasive effects of HL were observed at low concentrations (100, 150 and 200 μM) without growth-inhibition under IC 50 values (238 μM) [25].

Inhibitory effects of HL on migration
To examine whether the anti-invasive effect of HL is associated with its suppression of the cell motility, the effect of HL on the migration capability of cells was analyzed by using scratch wound assay. The confluent monolayer of A549 cells was scratch-wounded with 200 μl sterile pipette tips and post-incubated for a further 19 h in the absence (control) or presence of HL. The results are shown in Figure 6. HL inhibited the migration of A549 compared with control cells. The antimigrate effects of HL were observed at low concentrations (100 μM) without growth-inhibition under IC 50 values (238 μM) [25] and within the doubling time (22 h) of A549 cells in ATCC protocol. Ceramide activates protein phosphatase 2A (PP2A) [33], which is known to negatively regulate the phosphorylated-Akt (p-Akt) [34] and then Akt regulates actin organization and cell motility [35]. It has been reported that treatment of A549 cells with HL inhibited phosphorylation of Akt on the basis of immunocytochemistry using flow cytometer, and downregulation of p-Akt by HL could be related to blocking the actin cytoskeleton dynamics such as filopodia formation [26]. Thus, these results suggest that HL could induce ceramide generation, downregulate p-Akt and block the actin cytoskeleton, which inhibit migration of A549 cells.

Conclusion
We clearly demonstrated the ceramide generation and invasive inhibition by HL on A549 cells. The noteworthy aspects in this study are as follows: (a) Ceramide generation for A549 cells treated with HL was observed through the activation N-SMase. (b) Sphingomyelin in A549 cells after the treatment with HL decreased. (c) Anti-invasive effects of HL for A549 cells were obtained through the inhibition of MT1-MMP/MMP14. (d) Inhibitory effects of HL on migration of A549 cells was obtained using scratch wound assay. The results in this study could be advantageous in chemotherapy for patients with NSCLC in clinical applications.