WO2013107839A2 - Phospholipid compounds for use in skin cancer treatment - Google Patents

Abstract

The present invention relates to phospholipid compounds of formula I wherein R¹ is selected from the group consisting of: -CO(CH₂)_pCHO and -CO(CH₂)_pCOOH, wherein p = 1-7, and R² is selected from the group consisting of: -CO(CH₂)_nCH₃ and -(CH₂)_mCH₃, wherein n = 8-16 and m = 8-20, for use in the treatment of skin cancer and precancerous skin lesions.

Description

Phospholipid compounds for use in skin cancer treatment

The present invention relates to phospholipid compounds for use in the treatment of skin cancer and precancerous skin lesions as well as to pharmaceutical compositions comprising these compounds.

Cancer is a complex disease characterized by genetic mutations that lead to uncontrolled cell growth. Cancerous cells are present in all organisms and under normal circumstances their excessive growth is tightly regulated by various physiological factors. One such regulatory process is apoptosis or programmed cell death.

Apoptosis is an important mechanism in both development and homeostasis in adult tissues for the removal of either superfluous, infected, transformed or damaged cells by activation of an intrinsic suicide program. It is an active process with unique morphological and biochemical features including cell shrinkage, membrane blebbing, chromatin condensation with subsequent internucleosomal fragmentation of DNA, organelle relocalization, formation of discrete membrane enclosed vesicles (apoptotic bodies), and finally cell fragmentation without leakage of cytosolic macromolecules.

In contrast, necrosis is a passive process characterized by an increase in cell volume, swelling of the mitochondria and loss of membrane integrity, resulting in cell lysis and spillage of cellular contents into the environment, that generally leads to an inflammatory response.

Skin neoplasms also known as "skin cancer" are skin growths of melanocytic and non- melanocytic cells with differing causes and varying degrees of malignancy. The three most common malignant skin cancers are basal cell carcinoma, squamous cell carcinoma, and melanoma, each of which is named after the type of skin cell from which it arises.

Melanoma is a cancer that develops in melanocytes, the pigment cells present in the skin, or presumably in a melanoma stem cell. It can be more serious than the other forms of skin cancer because it may spread to other parts of the body (metastasize) resulting in death. At advanced stage melanoma survival rates are poorer than for non-melanoma skin cancer, however, melanomas diagnosed at an early stage are more accessible to complete removal by surgery and thus survival rates are high. The treatment of melanoma ususally comprises surgical removal of the tumor, adjuvant treatment, chemo- and immunotherapy, or radiation therapy. In the case of disease that has spread (metastasized), further surgical procedures or chemotherapy may be required.

Additional treatment approaches include chemoimmunotherapy, targeted therapy (e.g., monoclonal antibody therapy) and vaccine therapy.

Chemotherapy has long been a mainstay for treating metastatic melanoma, that is, melanoma that has spread beyond its site of origin. Although there is no clear consensus among clinicians about the most effective use of these drugs, chemotherapy does help some people and so different types are available, including dacarbazine, paclitaxel, cisplatin and carmustine. Chemotherapy as an overall strategy is not very effective in treating melanoma. Only 15% to 20% of patients respond to chemotherapy, it typically only works for less than a year, and it has little to no effect on survival time, not to mention the side effects.

Moreover, it is difficult to devise effective treatment regimen because the chemotherapeutics presently available do not act equally on all melanoma due to their genetic heterogeneity.

Basal cell carcinoma (BCC) is the most common type of skin cancer. It rarely metastasizes or kills. However, because it can cause significant destruction and disfigurement by invading surrounding tissues, it is a malignant tumor. Statistically, approximately 3 out of 10

Caucasians may develop a basal-cell cancer within their lifetime. In 80 percent of all cases, basal-cell cancers are found on the head and neck. There appears to be an increase in the incidence of basal-cell cancer of the trunk (torso) in recent years.

Among the methods usually employed in the treatment of BCC are standard surgical excision, Mohs micro graphic surgery, electrodesiccation and curettage, cryosurgery, laser surgery, radiation therapy, topical chemotherapy with fluorouracil and photodynamic therapy.

Squamous cell carcinoma (SCC) is a histologically distinct form of cancer. It results from the uncontrolled expansion of malignant cells originating from epithelium, or showing particular cytological or tissue architectural characteristics of squamous cell differentiation, such as the presence of keratin, monofilament bundles, and/or desmosomesX. Squamous cell carcinoma is one of the most common cancers in humans and other animals, and usually arises from mutated ectodermal or endodermal cells lining body cavities. Therefore, it can develop in a large number of organs and tissues, including the skin, lips, mouth, esophagus, urinary bladder, prostate, lung, vagina, and cervix, among others. Despite the common name, squamous cell carcinomas arising in different body sites can show tremendous differences in their presenting signs and symptoms, natural history, prognosis, and response to treatment. Treatment methods are similar to those of BCC. In addition chemotherapy is an option for advanced disease.

Certain cutaneous lesions of melanocytic and non-melanocytic origin serve both as precursors of skin cancer and as markers for increased risk. A precancerous skin lesion in connection with non-melanocytic skin cancers is actinic keratosis. Actinic keratoses are bona fide known as already representing histopathologically a carcinoma in situ. Clinically, these keratoses manifest as rough, scaly, erythematous patches on chronically sun-exposed surfaces. Another precancerous skin lesion and the second precursor for invasive non- melanocytic forms of skin cancer is Bowen's disease (squamous cell carcinoma in situ). Still another precancerous skin lesion that is the most important precursor/marker for melanoma is the clinically atypical mole (CAT) which has been observed to evolve into cutaneous melanoma.

Treatments of actinic keratosis currently encompass topical agents such as fluorouracil, imiquimod cream, diclofenac sodium gel and trichloroacetic acid, as well as cryosurgery, curettage, dermabrasion, shave excision, photodynamic therapy and carbon dioxide laser.

As actinic keratoses arise within UV-irradiated skin, clearance of these lesions will not sufficiently suppress the occurrence of new lesions in adjacent skin. The term 'field cancerisation' was coined by oncologists to adjust for a need of treating not only overt actinic keratosis, presenting just the tip of the iceberg, but rather vigorously the entire area of skin affected by a carcinogen such as UV.

Therefore a need for therapeutic compounds exists, which allow to bypass the genetic heterogeneity of tumor cells and are generally applicable for the treatment of various types of skin cancer such as primary melanoma, metastatic melanoma, BCC and SCC, as well as precancerous skin lesions, i.e., skin cancer precursors representing a carcinoma in situ. In the latter case, in particular, treatments which focus on the topical application of effective agents onto the affected sites have gained in importance.

Scientific research has recently focused on phospholipid oxidation products. It has been found that homologous oxidized phospholipids which are typically present in oxidatively modified low density protein induce different cellular responses like inflammation, proliferation or cell death. Thus, they selectively activate processes in vascular wall cells that may contribute to the pathogenesis of atherosclerosis as well as other chronic inflammatory diseases. Furthermore, they have been demonstrated to inhibit growth and induce apoptosis in vascular smooth muscle cells (Fruhwirt, G. O. et al., Biochimica et Biophysica Acta , 1761(9), 2006: 1060-1069).

WO 2008/074573 A discloses the topical use of the compound Edelfosine (l-octadecyl-2-O- methyl-OT-glycero-3-phosphocholine), a synthetic alkyl-lisophospholipid, for use in the treatment of cancer, in particular skin cancer.

It is the object of the present invention to provide compounds and pharmaceutical compositions for use in the treatment of skin cancer, in particular primary melanomas, which are not accessible to surgery, metastatic melanoma, and basal cell carcinoma and squamous cell carcinoma as well as precancerous skin lesions such as actinic keratosis.

This object is achieved by a phospholipid compound of formula I

wherein R is selected from the group consisting of:

-CO(CH₂)_pCHO and

-CO(CH₂)_pCOOH, wherein p = 1-7,

and R is selected from the group consisting of:

-CO(CH₂)_nCH₃ and

-(CH₂)_mCH₃, wherein n = 8-16 and m = 8-20.

The phospholipids of formula I have been found to induce apoptosis in skin cancer cells of various types and thereby provide potent pharmaceutical ingredients for the treatment of skin cancer and precancerous skin lesions.

According to a preferred embodiment, in the compound of formula I, R is -CO(CH₂)_nCH . Among the compounds of this group the phospholipid l-palmitoyl-2-(5-oxovaleroyl)-5^,w- glycero-3-phosphocholine (POVPC) is particularly preferred, i.e., a compound in which R¹ is -CO(CH₂)_pCHO, p is 3 and n is 14. Another particularly preferred compound of this group is l-palmitoyl-l-glutaroyl-^w-glycero-S-phosphocholine (PGPC), i.e., a compound in which R¹ is -CO(CH₂)_pCOOH, p is 3 and n is 14.

According to another preferred embodiment, in the compound of formula I, R is - (CH₂)_mCH₃. Among the compounds of this group the phospholipid l-0-hexadecyl-2-(5- oxovaleroy^-sw-glycero-S-phosphocholine (E-POVPC) is particularly preferred, i.e., a compound in which R¹ is -CO(CH₂)_pCHO, p is 3 and m is 15. Another particularly preferred compound of this group is l-0-hexadecyl-2-glutaroyl-s7i-glycero-3-phosphocholine (E- PGPC), i.e., a compound in which R¹ is -CO(CH₂)_pCOOH, p is 3 and m is 15.

According to a preferred embodiment, the phospholipid compound of formula I is used in the treatment of skin cancers selected from primary melanomas and metastatic melanoma. Advantageously, the phospholipid compound is used in the treatment of primary melanomas which are selected from the group consisting of lentigo maligna, lentigo maligna melanoma and primary melanomas which are not accessible to surgery.

According to another preferred embodiment, the phospholipid compound of formula I is used in the treatment of non-melanoma skin cancer, preferably basal-cell carcinoma or squamous cell carcinoma.

According to a further preferred embodiment, the phospholipid compound of formula I is used in the treatment of precancerous skin lesions, preferably actinic keratosis.

According to another aspect of the present invention, the phospholipid compound of formula I is preferably used for the topical treatment of skin cancer and precancerous skin lesions.

The compounds of the present invention can also be advantageously applied in field cancerisation treatments, particularly after clearance of actinic keratosis lesions, in order to suppress or prevent the occurrence of new lesions.

The phospholipid compounds according to the invention are naturally produced in the human body and therefore hold promises in terms of therapeutic benefits, as they avoid the toxicity problems of synthetic compounds such as Edelfosine and can be easily metabolized by homogenous enzymes. Furthermore, while the cell death induced by the synthetic compound Edelfosine is mostly due to necrosis, the phospholipid compounds according to the invention have been shown to lead to cell death of skin cancer cells mainly by inducing apoptosis.

According to a further aspect, the invention provides a pharmaceutical composition for use in the treatment of skin cancer and precancerous skin lesions, comprising a phospholipid compound of formula I as defined above and a pharmaceutically acceptable carrier therefor. Preferably, the pharmaceutical composition is designed or adapted for use by topical application.

The present invention will be explained in more detail by way of the following examples and the attached drawings:

Fig. 1 depicts the chemical structure of the oxidized phospholipids PGPC and POVPC, the ether-phospholipids E-PGPC and E-POVPC, and Edelfosine:

Fig. 2 illustrates the time-dependent hydrolysis of PGPC and POVPC under low serum conditions as shown by thin-layer chromatography. Oxidized phospholipids were incubated in RPMI-1640 culture medium supplemented with 0.1 % FBS for various time points. After incubation, lipids were extracted with organic solvents and lipids were separated by thin- layer chromatography. For separation of POVPC (RF=0.38) and PLPC (RF=0.26) from other phospholipids, a neutral mobile phase was used, whereas for separation of PGPC (RF=0.41) and PLPC (RF=0.30) an acidic mobile phase was applied (see methods). Under high serum conditions, hydrolysis of both oxPLs starts after 3 hours (data not shown).

However, under low serum conditions, PGPC is stable for at least 6 hours, whereas degradation of POVPC begins after 3 hours and is not fully finished after 6 hours of incubation. For all experiments, a significant amount of oxPLs remains intact over the incubation time period.

Fig. 3(a-j) shows the results of MTT viability assays of melanocytes and melanoma cells stimulated with PGPC and POVPC. Cells were incubated with the indicated concentrations of oxPLs, PLPC or POPC in Melanocyte Growth Medium or RPMI-1640 with 0.1 % FBS for 2h, 12h and 20h. POPC (negative control) and PLPC (positive control) were used as native reference phospholipids. All cell lines were analyzed by the MTT photometric assay to determine the effect of oxPLs on the viability of the cells in a concentration- and time dependent manner. High absorbance values correspond to high viability. POPC does not affect cell viability in any tested cell line, whereas PGPC, POVPC and PLPC show to be cytotoxic in a concentration-dependent manner. Longer incubations times lead to similar decreases in cell viability, compared to short incubation times. Ethanol as a negative control does not show any effect (data not shown). Results were obtained from 2 replicates of at least three independent experiments and values represent means + S.D.

Fig. 4(A/B) shows the results of cell death of cultured human melanocytes and melanoma cells induced by oxidized phospholipids PGPC and POVPC. Cells were incubated with the indicated concentrations of oxPLs for 6 hours in melanocyte growth media or RPMI-1640 supplemented with 0.1 % FBS. Control cells were treated with H₂0₂ (positive control for necrosis) or 1 % (v/v) EtOH (negative control). All cell lines were analyzed by flow cytometry to determine the percentage of necrotic and apoptotic cells. Cells stained by green fluorescent AlexaFluor488^® Annexin V, which binds to phosphatidylserine at the cell's outer plasma membrane leaflet, but not by red fluorescent PI, which is membrane impermeant and thus does not stain intact cells, were defined to be apoptotic. Cells stained by PI were defined as necrotic cells. Panel A: Neither PGPC nor POVPC induce necrosis under the indicated conditions. Panel B: POVPC is a stronger inducer of apoptosis than PGPC under the indicated conditions. POVPC induces phosphatidylserine exposure, a sign of apoptosis, in all tested cell lines in a concentration dependent manner, with FOM melanocytes showing a significantly lower rate of apoptosis compared to melanoma cell lines. In contrast, only in SBcl2 cells apoptosis can be induced by stimulation with PGPC. Results were obtained from 2 replicates of at least two independent experiments and values represent means + S.D. *P < 0.05 compared with control.

Fig. 5(A-F) illustrates the effects of POVPC and PGPC on acid sphingomyelinase activity in human melanocytes and human melanoma cell lines. Human melanocytes (FOM) and melanoma cell lines of different stages were incubated with 50 μΜ oxPL or 1 % (v/v) EtOH as a control for 5 min and 15 min. Cells were harvested and lysed in lysis buffer for assaying acid sphingomyelinase activity (see Methods). Enzyme activities were determined as described previously (7) using NBD-labelled sphingomyelin as substrate. Formed NBD- ceramide was separated from the remaining substrate by TLC on silica gel and analysed using a CCD-camera. The ratio of formed NBD-ceramide to NBD-sphingomyelin was determined and expressed as fold of control at time point zero. Data are means + S. D. (n > 4). A: representative TLC for the cell line SBcl2, incubated with 50 μΜ oxPLs or EtOH (1 % v/v) as a control for the indicated times, B: activation of acid sphingomyelinase in FOM melanocytes after stimulation of the cells with the oxPLs, C-F: results obtained for the cell lines SBcl2, WM35, WM9 and WM164, respectively. *P < 0.05 compared with control. **P < 0.005 compared with control. Fig. 6(A-E) illustrates the oxidized phospholipid-dependent formation of different ceramide species and sphingomyelin species in melanocytes and melanoma cells. Cells were incubated with 50 μΜ oxPLs for different time points, lipids were extracted and ceramide and sphingomyelin species were analysed as described in material and methods. For the determination of lipid to protein ratios, lipid amounts were referenced to an internal standard. Panel A-E: Lipid to protein ratios of different ceramide species and sphingomyelin species after stimulation with 50 μΜ POVPC for 15 minutes or 6 hours. A: Fom

melanocytes, B: primary melanoma cell line SBcl2 C: primary melanoma cell line WM35 D: metastatic melanoma cell line WM9 E: metastatic melanoma cell line WM164.

Fig. 7(A-D) shows the result of the MTT viability assay of B16 mouse melanoma cells stimulated with different oxidized phospholipids. Cells were incubated with different concentrations of PGPC and POVPC for 2 hours, 14 hour or 24 hours in DMEM

supplemented with 0.1 % FBS. POPC was used as a native reference phospholipid, PLPC was used as a positive control for the induction of cell death. Cells were analyzed by the MTT photometric assay to determine the effect of different lipids on the cell viability in a concentration- and time-dependent manner. POPC does not affect cell viability at any time point. Both oxidized phospholipids reduce cell viability in a concentration- and time- dependent manner. Results were obtained from 2 replicates of at least three independent experiments and values represent means + S.D.

Fig. 8(A-C) shows the results of cell death analysis of B16 mouse melanoma cells induced by oxidized phospholipids, oxidized ether phospholipids and Edelfosine. Cells were incubated with different concentrations (25 μΜ or 50 μΜ) of the indicated lipids for 6 hours in DMEM (with 0.1 % FBS). Control cells were incubated with EtOH (1 % v/v) as a negative control, 10 μΜ STS (positive control for apoptosis) or 10 mM H₂0₂ as a positive control for necrosis. All cells were analyzed by flow cytometry (for details see Material and Methods) to determine the percentage of apoptotic and necrotic cells. Cells stained by red PI were defined as necrotic cells. Cells stained by green AlexaFluor488® Annexin V, but not by PI, which is membrane impermeant and thus does not stain intact cells, were defined as apoptotic cells. Panel A: necrosis induced by different lipids in B16 mouse melanoma cells. Panel B: amount of apoptotic cells after incubation with the indicated lipids for 6 hours. Panel C: morphological changes of cells incubated with the indicated lipids and controls for 6 hours. Results were obtained from 2 replicates of at least three independent experiments and values represent means + S.D. *P < 0.05 compared with control. **P < 0.0001 compared with control. Fig. 9 shows the effects of oxPL on acid sphingomyelinase activity in B16 melanoma cells. Murine B16-BL6 melanoma cells were incubated with 25 μΜ oxPL or 1% (v/v) EtOH as a control in DMEM (0.1% FBS) without Phenol red for the indicated times. Cells were harvested and lysed in acid lysis buffer for assaying acid sphingomyelinase activity according to the method of Loidl et al. (7). Enzyme activities were determined using

NBD-labelled sphingomyelin as a substrate. The fluorescent NBD-ceramide product was separated from the remaining sphingomyelin substrate by thin layer chromatography on silica plates using CHCl₃:MeOH:H₂0 (65:24:4 per vol.) as solvent. Fluorescence intensities were analysed using a CCD camera, and the ratio of NBD-ceramide to total NBD lipid (Cer + SM) was determined. Results represent means + S.D. (*P < 0.05 compared with control, n > 3).

Fig. lO(A-D) illustrates the effect of oxPL on ceramide and sphingomyelin patterns in B16 murine melanoma cells. Cells were incubated with 25 μΜ PGPC or POVPC or 1% EtOH (v/v) (negative control) for 6 hours in DMEM supplemented with 0.1% FBS, lipids were extracted and ceramide and sphingomyelin species were analysed as described in Material and methods. Panel A+C: Relative amounts of ceramide and sphingomyelin species. Panel B+D: Total ceramide and sphingomyelin contents. Values represent means + S.D. (*P < 0.05 compared with control, n = 3).

Fig. 1 l(A-C) gives the effects of oxPL on migration of cultured murine B16 melanoma cells. Cultured B16-BL6 melanoma cells were analysed for their potential to migrate into a cell-free scratch region under the influence of 5 μΜ oxPL (see Materials & Methods). Panel A: Microscopy images of cells after 0 h, 10 h and 30 h incubation with oxPL. The % width of the cell-free scratches relative to control cells are expressed as reciprocal values (% closure) in panels B and C. Panel B: Effects of 5 μΜ PGPC or POVPC or 1% (v/v) EtOH (negative control). Panel C: Effects of 5 μΜ oxidized ether-phospholipids E-PGPC or E-POVPC, in comparison to untreated control cells. Data are expressed as means + S.D. (n > 3).

Fig. 12(A-C) shows the effects of oxPL on cell morphology/integrity of HaCaT

keratinocytes and squamous carcinoma cell lines. Cells were seeded into 24- well plates in full growth medium and allowed to attach to the surface over night. Following several washing steps to remove floating cells, cells were incubated with different concentrations of PGPC or POVPC in medium containing 0.1 % FBS. Control cells were incubated with EtOH (1% v/v) or medium (negative controls) or with H₂0₂ (necrosis control). Microscopic images were taken after the indicated incubation times. Representative results are shown for HaCaT keratinocytes (Panel A), SCC12 cells (Panel B) and SCC13 cells (Panel C).

Fig. 13(A/B) illustrates the effect of oxidized phospholipids and ether phospholipids on the viability of HaCaT keratinocytes and SCC13 squamous carcinoma cells after 2 hours.

HaCaT keratinocytes (Panel A) and SCC13 cells (Panel B) were incubated with different lipid concentrations in low serum medium (0.1% FCS) for 2 hours (PGPC , POVPC , E-PGPC , E-POVPC, PLPC , POPC ). Media containing EtOH and DMSO (1% (v/v)) were used as negative controls. H₂0₂ and STS represent positive controls for induction of necrosis and apoptosis, respectively. Cell viabilities were determined using the MTT viability assay. Viability of control cells (EtOH) was set to 100% and all other values represent % viabilities relative to the control. Results were obtained from three or more independent experiments and values represent means +/- SD.

Fig. 14(A/B) gives the effects of oxPL on apoptosis and necrosis of cancer cells. HaCaT cells and SCC13 cells were incubated with different concentrations of oxPL for 6 h in low serum medium (0.1 % FCS). Control cells were incubated with medium containing 1% (v/v) EtOH. Cells were incubated with 20 μΜ STS or 30 mM H₂0₂ as positive controls for apoptosis and necrosis, respectively. After stimulation, cells were harvested and, after staining with propidium iodide (PI) and Annexin V, analysed by flow cytometry. Intact cells were unstained. Cells stained by PI or both dyes were considered necrotic. Cells stained by Annexin V were considered apoptotic. Panel A: Apoptotic cells. No apoptosis can be detected in HaCaT keratinocytes, whereas all oxPL induce apoptosis in a concentration- and lipid-dependent manner in SCC13 cells. Panel B: Necrotic cells. In HaCaT cells, oxPL lead to a slight increase of necrosis, whereas no necrosis can be found in SCC13 cells. Results are expressed as means +/- SD (n > 3).

Fig. 15 shows the effects of oxPL on total ceramide and sphingomyelin levels in SCC13 cells. SCC13 cells were incubated with 50 μΜ PGPC or POVPC or 1% EtOH (v/v) (negative control) in RPMT1640 medium (0.1% FCS) for 6 hours. Cells were harvested, lipids were extracted, and total amounts of ceramide and sphingomyelin were determined as described. PGPC does not change total ceramide and sphingomyelin levels in SCC13 cells. In contrast, POVPC leads to a significant increase in total ceramide and sphingomyelin. Data are expressed as means +/- SD. Significances were determined by Student's t-test (two tailed, unpaired). * P < 0.05 compared with control. Fig. 16 illustrates the effect of oxPL on acid sphingomyelinase activity in SCC13 cells. SCC13 cells were incubated with RPMI-1640 media containing 50 μΜ oxPL or 1% EtOH (v/v) as negative control. Cells were harvested, lysed and acid sphingomyelinase activities were determined. Data are expressed as % activity of unstimulated cells. Stimulation of aSMase by PGPC is significantly increased between 15 and 30 min, whereas POVPC shows hardly any effect. Values are expressed as means + SD.* P < 0.05 compared with control (n > 3).

Fig. 17(A/B) illustrates the influence of oxPL on migration of HaCaT cells and SCC13 cells. Cultured HaCaT keratinocytes (Panel A) and SCC13 carcinoma cells (Panel B) were analysed for their potential to migrate into a cell-free zone under the influence of oxPL. For this purpose, cells were incubated with 5 or 10 μΜ oxPL and microscopic images were taken from identical areas within one well after the indicated incubation times. The width of the cell-free zone was measured using ImageJ software and the extent of migration was calculated as percentage of the initial width of the gap. Results represent means +/- S.D. (n > 3).

Examples

Abbreviations: aSMase acid sphingomyelinase

Cer ceramide

DMEM Dulbeccos modified Eagle medium

Edelfosine l-octadecyl^-O-methyl-sw-glycero-S-phosphocholine

E-PGPC l-O-hexadecyl^-glutaroyl-sw-glycero-S-phosphocholine

E-POVPC l-0-hexadecyl-2-(5-oxovaleroyl)-s7i-glycero-3-phosphocholine

EtOH ethanol

FACS fluorescence activated cell sorting

FBS/FCS fetal bovine serum

HaCaT keratinocytes human adult low calcium high temperature keratinocytes

HPLC high-performance liquid chromatography

MGM Melanocyte Growth Medium

MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NBD N-7-nitrobenz-2-oxa- 1 ,3-diazol

NBD-Cer N-7-nitrobenz-2-oxa- 1 ,3-diazol-ceramide

NBD-SM N-7-nitrobenz-2-oxa- 1 ,3-diazol-sphingomyelin oxLDL oxidized low-density lipoprotein

oxPL oxidized phospholipid

PGPC l-palmitoyl-2-glutaroyl-5'w-glycero-3-phosphocholine

PI propidium iodide

PLPC l-palmitoyl-sw-glycero-S-phosphocholine

POPC l-palmitoyl-2-oleoyl-5'w-glycero-3-phosphocholine

POVPC l-palmitoyl-2-(5-oxovaleroyl)-5'w-glycero-3-phosphocholine

PS phosphatidylserine

RPMI medium Roswell Park Memorial Institute medium

SCC squamous cell carcinoma

SFM serum-free medium

SM sphingomyelin

TLC thin layer chromatography

Materials:

PGPC and POVPC were either synthesized in the inventor' s laboratory as previously described (1) or were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). POPC was synthesized according to the method of Hermetter et al. (2). PLPC was acquired from Bachem (Bubendorf, Switzerland). Edelfosine, E-PGPC and E-POVPC were synthesized in the inventors' laboratory (12). Tissue culture dishes and flasks were obtained from Sarstedt (Niiraibrecht, Germany) or Greiner (Kremsmiinster, Austria). DMEM media with or without phenol red, RPMI- 1640 media with or without Phenol red, Fetal Bovine Serum (FBS = Fetal Calf Serum FCS) and trypsin were purchased from Gibco (Carlsbad, CA), Melanocyte Growth Medium with or without Phenol red was purchased from Promo Cell (Heidelberg, Germany). Phosphate buffered saline (PBS) and all other supplements for cell culture were acquired from PAA Laboratories (Linz, Austria), unless otherwise indicated. Vybrant® MTT Cell proliferation Assay kit (V- 13154), Vybrant® apoptosis assay kit#2 (V- 132451) and staurosporine were from Invitrogen (Leek, Netherlands). Flow cytometry fluids and FACS tubes were from BD bioscience (Heidelberg, Germany). Cell culture-inserts for self- insertion were acquired from Ibidi GmbH (Munich, Germany). Organic solvents and all other standard chemicals were obtained from Carl Roth (Karlsruhe, Germany) or Sigma- Aldrich (Vienna, Austria), unless otherwise indicated.

A) Human melanoma tumor cells

The uptake and the toxic effects of oxidized phospholipids on human melanocytes and 4 different melanoma cell lines (primary melanoma cell lines SBcl2 and WM35, metastatic melanoma cell lines WM9 and WM164) were investigated. For this purpose two chemically defined oxidized phospholipids (PGPC and POVPC) were used, which are oxidation products of phosphatidylcholine. Both oxPLs contain a single hydrophobic fatty acid at the sn-l position and only differ in their short polar fatty acyl chains in position 2 of glycerol (Fig. 1). In case of PGPC, the residue at the sn-2 position is a carboxylic acid; in contrast, POVPC contains a highly reactive aldehyde group that allows the molecule to interact chemically with its targets by undergoing Schiff base formation.

Cell Culture and incubation with oxidized phospholipids

Human primary melanoma cell lines (SBcl2, WM35) and metastatic melanoma cell lines (WM9, WM164) were cultured in RPMI-1640 supplemented with 2% FBS, 200 units/ml penicillin/streptomycin and 4 mM L-glutamine. Human melanocytes FOM, derived from human foreskin, were cultured in Melanocyte Growth Medium. All cells were routinely grown at 37°C in humidified C0₂ (5%) atmosphere. For all experiments, ethanolic solutions of lipids containing the indicated μΜ concentration of lipid were prepared using the ethanol injection method (3). The final concentration of EtOH was always kept below 1% (v/v). Incubation medium for all experiments was medium containing 0.1% FBS without Phenol red unless otherwise indicated. Control experiments were carried out with incubation medium containing 0.1% FBS and the same amount of EtOH but without lipids.

Example 1 : PGPC and POVPC are converted to lysophosphatidylcholine (PLPC) by fetal bovine serum

In previous studies the cytotoxic effects of oxidized phospholipids on vascular smooth muscle cells in DMEM culture medium were abolished under high serum conditions (10% FBS) (4). In order to find out if the oxidized phospholipids PGPC and POVPC are stable in the used culture media under different serum conditions, the oxPLs were incubated in RPMI- 1640 growth media supplemented with 0.1% FBS used in all experiments and the results were compared with the stability of the oxPLs when incubated in media supplemented with 2% FBS. The oxidized phospholipids were added to the media using ethanol injection method and incubated for varying time points at 37°C. PGPC, POVPC, serum lipids and the formed degradation product (PLPC) were subsequently separated by thin layer

chromatography on silica plates. At low serum conditions (2% FBS), the hydrolytic cleavage of the oxidized sn-2 chain of both PGPC and POVPC starts after 3 hours of incubation and is not fully completed after 6 hours (data not shown). However, in media supplemented with 0,1% serum, PGPC is stable for at least 6 hours and no degradation products are detectable. In case of POVPC, hydrolysis starts after 3 hours, leading to the formation of PLPC (Fig. 2), but this degradation is not fully completed even after 20 hours of incubation (data not shown).

Method:

Stability of oxidized phospholipids in RPMI-1640 media supplemented with 0.1% or 2% FBS was determined as previously described (4). In detail, lipid dispersions containing 100 μΜ of PGPC or POVPC in media supplemented with varying concentrations of FBS were prepared and incubated at 37°C shaking (550 rpm) for different time points. After incubation, phospholipids were extracted with chloroform/methanol 2: 1 (v/v) and short intense mixing. Extraction was repeated once more, the organic phases were combined and removed under a gentle stream of nitrogen. The lipids were dissolved in

chloroform/methanol 2: 1 (v/v) and analyzed by thin layer chromatography on silica plates. For the separation of POVPC and its degradation product PLPC, the mobile phase was chloroform/methanol/water 30/50/10 (v/v/v). PGPC and PLPC were separated using an acidic mobile phase containing chloroform/methanol/acetone/glacial acetic acid/water 20/40/10/10/10 (v/v/v/v/v). After separation, lipid spots were detected using molybdenum blue reagent which specifically stains phospholipids (5). Different phospholipids were identified by comparison with pure reference compounds.

Example 2: Oxidized phospholipids induce cell death in human melanoma cells

In order to determine the effects of the oxidized phospholipids PGPC and POVPC on cell viability, a MTT viability assay was used. Human melanocytes (FOM), human primary melanoma cells (SBcl2, WM35) and human metastatic melanoma cells (WM9, WM164) were incubated with different concentrations of the oxPLs (from 5 to 350 μΜ) for 2 hours, 12 hours and 20 hours in MGM or in RPMI-1640 containing 0.1% FBS. POPC was used as a native reference phospholipid, and PLPC as a positive control for the induction of cell death (12). All cell lines display the same tendencies depending on concentration and time, but they show different sensitivities to treatment with PGPC and POVPC. In all cell lines incubation with the oxidized phospholipids induces a concentration dependent increase in cell death already after 2 hours. POPC does not affect cell viability in any tested cell line, whereas PGPC, POVPC and PLPC show to be cytotoxic in a concentration-dependent manner. Longer incubation times lead to similar decreases in cell viability, compared to short incubation times. Ethanol as a negative control does not show any effect (data not shown) (Figure 3). In human FOM melanocytes, the effect of PGPC and POVPC is strongest after two hours of incubation, and reduction of cell viability decreases with longer incubation times. In contrast, in all melanoma cell lines two hours of stimulation with PGPC or POVPC are sufficient to reduce cell viability, but the strongest effect is observable after 20 hours of stimulation. Regarding concentration dependency of the decrease of cell viability, already low concentrations of both oxidized phospholipids (50 μΜ) are sufficient to reduce cell viability in all cell lines, with the exception of the primary melanoma cell line WM35. This cell line is less sensitive when incubated with PGPC, and even higher concentrations are needed when incubated with POVPC to show the same cytotoxic effects. The non-oxidized phospholipid POPC does not affect cell viability in all tested cell lines independent of the incubation times and the concentrations that are used. Raising the serum concentration of the incubation medium from 0.1% FBS to 2% FBS diminishes the effects of PGPC and POVPC but does not completely abolish them (data not shown) which can be explained by the degradation of PGPC and POVPC by serum after longer incubation times.

Method:

MTT viability assay

To determine the cytotoxic effect of PGPC and POVPC in human melanocytes and melanoma cells, the Vybrant® MTT Cell proliferation Assay kit was used according to the manufacturer's recommendations. The MTT assay involves the conversion of the water soluble MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to an insoluble formazan by viable cells. The formazan is then solubilised, and the concentration determined by measuring the optical density at 595 nm. The protocol was optimized for each cell line according to cell number, MTT concentration and incubation times. In brief, cells were seeded in a 96 well plate and allowed to grow to 80% confluency in normal growth medium. The medium was replaced with the lipid dispersion at the desired concentration (between 5 μΜ and 350 μΜ) or control substances (1% EtOH (v/v), 2.5 mM H₂0₂, 10 μΜ

staurosporine) for 2 hours, 12 hours or 20 hours. After incubation, the lipid containing medium was replaced by 100 μΐ fresh medium, and 10 μΐ MTT (2.5 mg/ml) were added prior to 4 h incubation at 37°C. Subsequently 100 μΐ 10% SDS (w/v) in 0.01% HC1 (v/v) were added and incubated for 4 h under the same conditions. The mixture was resuspended carefully and optical density was detected at 595 nm using an Anthos plate reader driven by WinRead 2.3 software.

Example 3: Oxidized phospholipids induce apoptosis in human melanoma cells under low serum conditions

MTT experiments showed that incubation of human melanocytes and melanoma cells with oxidized phospholipids lead to a decrease in cell viability already 2 hours after stimulation (see Fig. 3). To find out if this effect is due to an increase in cell death, and whether this is caused by necrosis or apoptosis, the occurrence of phosphatidylserine (PS) at the outer leaflet of the plasma membrane, which is a typical sign of apoptosis, was analyzed by flow cytometry. Cells stained by green fluorescent AlexaFluor488^® Annexin V, which binds to PS at the cell's outer plasma membrane leaflet, but not by red fluorescent PI, which does not permeate the membrane and thus does not stain intact cells, were defined to be apoptotic. Cells stained by PI were defined as necrotic cells. FOM melanocytes, primary melanoma cells (SBcl2, WM35) and metastatic melanoma cells (WM9, WM164) were incubated in the presence or the absence of PGPC or POVPC (25 μΜ, 50 μΜ) for 6 hours and the percentage of apoptotic and necrotic cells compared to untreated control cells was analyzed. H₂0₂ was used as a positive control for necrosis. In all tested cell lines, both oxidized phospholipids cause an increase in cell death by apoptosis, but not by necrosis (Fig. 4, panel A). However, the sensitivity of the cell lines towards treatment with the oxPLs is dependent on which oxidized phospholipid is used and differs widely between the cell lines (Fig. 4, panel B). POVPC is a stronger inducer of apoptosis than PGPC under the indicated conditions.

POVPC induces phosphatidylserine exposure, a sign of apoptosis, in all tested cell lines in a concentration dependent manner, with FOM melanocytes showing a significantly lower rate of apoptosis compared to melanoma cell lines. The aldehyde-containing lipid POVPC is more cytotoxic than PGPC at low concentrations of the oxPLs. When treated with 25 μΜ or 50 μΜ PGPC, only in SBcl2 cells the number of apoptotic cells increases. In contrast, all other cell lines are unaffected by the stimulation with low concentrations of PGPC. However, when treated with POVPC (50 μΜ) all cell lines show an increase in phosphatidylserine exposure, with healthy FOM melanocytes being least affected by the oxPL and the metastatic cell lines WM9 and WM164 being most sensitive compared to the control cells. Again, SBcl2 cells were highly sensitive towards treatment with 25 μΜ of POVPC, while all other cell lines did not show significant increase in cell death at this concentration.

Method:

Flow cytometric analysis of apoptotic and necrotic cells

For determination of apoptotic and necrotic cell populations after treatment of cells with oxPLs, the Vybrant® apoptosis assay kit#2 was used according to a slightly modified manufacturer's protocol. Cells were seeded into 24- well plates and allowed to reach 80% confluency. Cells were treated with 400 μΐ lipid dispersion in incubation medium (25 μΜ or 50 μΜ PGPC or POVPC) for 6 hours. Incubation with 1 vol.% EtOH in incubation medium was used as a negative control, H₂0₂ was used as a positive control for necrosis. After the incubation period, the supernatant was collected, cells were harvested using accutase, wells were washed twice to collect any remaining cells and all fractions were combined. After centrifugation, cells were washed with PBS containing 2 mg/ml glucose and resuspended in 200 μΐ Annexin Binding Buffer. 100 μΐ of the cell suspension was mixed with 5 μΐ

AlexaFluor®488 Annexin V, 5.5 μΐ of an aqueous solution of PI (final concentration 1 mg/ml) and incubated in the dark for 15 minutes at room temperature. Subsequently samples were diluted with 400 μΐ PBS containing 2 mg/ml glucose, gently mixed and kept on ice until analysis. Stained samples were analyzed immediately using a FACS Calibur flow cytometer (BD Bioscience, NJ), measuring the green fluorescence emission at 530 nm and the red fluorescence emission above 575 nm upon excitation at 488 nm. Populations were separated into three groups: apoptotic cells were only stained by green fluorescent

AlexaFluor®488 Annexin V due to phosphatidylserine at the cell membrane's outer leaflet, but did not incorporate PI; necrotic cells were either red fluorescent or double stained; live cells were unstained. Each experiment was carried out twice and each sample was done in parallel. The percentage of apoptotic cells was calculated using WinMDI 2.8 software package.

Example 4: Activation of acid sphingomyelinase by POVPC and PGPC

Sphingomyelinases are central elements in stress-induced signal transduction. They catalyze the hydrolysis of sphingomyelin, thus generating the second messenger ceramide which is a key upstream component of apoptotic signalling. In previous studies it has been shown that PGPC and POVPC activate the sphingomyelinase pathway, particularly acid

sphingomyelinase, in vascular smooth muscle cells (11). To investigate the effect of PGPC and POVPC on the activation of aSMase in human melanocytes and melanoma cells, the inventors used fluorescent NBD-sphingomyelin as a substrate (7). Fig. 5 shows the activation of acid sphingomyelinase as a consequence of stimulation by 50 μΜ POVPC or PGPC after 5 minutes and 15 minutes incubation time.

In FOM melanocytes, POVPC does not lead to a significant increase of acid

sphingomyelinase within 15 minutes of stimulation, and PGPC only shows minor effects on the activation level of this enzyme (Figure 5B). In contrast, stimulation of the melanoma cell induces a substantial increase in aSMase activity, but this activation is dependent on the cell line, the oxPL used and the incubation time.

All four tested melanoma cell lines show a significant increase of aSMase activity after 15 minutes when stimulated with PGPC or POVPC, but the extent of activation differs widely between the cell lines, with SBcl2 cells (Figure 5C) and WM9 cells (Figure 5E) showing the highest activation of aSMase. Incubation of WM35 cells (Figure 5D) and WM164 cells (Figure 5F) with oxPLs leads to a significant but less substantial activation of aSMase. In all cell lines, aSMase levels return to control levels after 60 min of stimulation (data not shown).

Methods:

Determination of acid sphingomyelinase activity

Human melanocytes FOM, primary melanoma cells (SBcl2, WM35) and metastatic melanoma cells (WM9, WM164) were grown on 60 mm Petri dishes to 80% confluency in RPMI-1640 (supplemented with 2% FBS) or MGM. Prior to stimulation with the oxPLs, cells were washed twice with media containing 0.1% FBS to remove excess FBS. Cells were subsequently incubated with 2 ml of a 50 μΜ oxPL dispersion in RPMI-1640 (0.1% FBS) or MGM, or 1 vol.% EtOH (negative control) for 5 minutes and 15 minutes. After treatment of the cells with the respective compounds, cells were washed with ice-cold PBS, scraped, harvested by centrifugation (1500 rpm, 10 min, 4°C) and lysed by incubation with acid lysis buffer (250 mM sodium-acetate, 0.2% Triton X-100, pH 5.0). The protein content of the samples was determined by the method of Bradford (6) and aliquots of cell lysate containing 15 μg of protein were used for the determination of acid sphingomyelinase activity using a fluorescent sphingomyelin substrate as described previously (7). Results were obtained using thin layer chromatography (mobile phase was CHCl₃:MeOH:H₂0 65:25:4 v/v/v) and quantification of the labelled fluorescent sample spots was done with a charged coupled device camera (Herolab, Vienna) at an excitation wavelength of 365 nm using EasyWin software.

Example 5: Stimulation of melanoma cell lines with PGPC or POVPC leads to the formation of different ceramide species and sphingomyelin species

Acid sphingomyelinase assays showed, that upon stimulation of melanocytes and melanoma cells with the oxidized phospholipids POVPC and PGPC, there is an increase in aSMase activity that is dependent on the oxidized phospholipid, the incubation time and the cell line used. To investigate which ceramide species and sphingomyelin species are formed after treatment of the cells, the inventors compared the ceramide and sphingomyelin patterns of unstimulated cells with the patterns of cells stimulated with 50 μΜ POVPC after 15 minutes and 6 hours incubation time (for details see Material and Methods).

Figure 6 summarizes the oxPL dependent formation of different ceramide species and sphingomyelin species in melanocytes and melanoma cells. No significant differences between stimulated and unstimulated cells in ceramide composition and sphingomyelin species can be detected in FOM melanocytes (Figure 6A), primary melanoma cells WM35 (Figure 6C) and metastatic melanoma cells WM164 (Figure 6E). In contrast, CI 6:0 ceramide, CI 8:2 ceramide and C24:2 ceramide are significantly increased in both SBcl2 cells and WM9 cells 6 hours after incubation with POVPC (Figure 6B and Figure 6D). In addition, C24:0 ceramide and C24: l ceramide are also increased in SBcl2 cells, but not in the other cell lines. Remarkably, this finding is in line with the activation of acid

sphingomyelinase in SBcl2 cells and WM9 cells after incubation with POVPC.

Method:

Identification and quantification of ceramide and sphingomyelin species

All cell lines were grown in 100 mm Petri dishes to 80 % confluency in full growth media. Prior to stimulation with the oxPLs, cells were washed twice with media containing 0.1% FBS to remove excess FBS. Cells were subsequently incubated with either 4.5 ml of a 50 μΜ oxPL dispersion in RPMI-1640 (0.1% FBS) or in melanocyte growth media for 15 minutes or 6 hours. 1 vol.% EtOH in the respective medium was used as a negative control. After treatment, cells were washed with ice-cold PBS, scraped into PBS and harvested by centrifugation (1500 rpm, 5 min, 4°C). Cells were resuspended in 1 ml PBS and an aliquot of 100 μΐ was used for measuring the protein content of the samples. For this purpose, cells were harvested by centrifugation (640 g, 5 min, 4°C) and lysed in 70 μΐ of neutral lysis buffer for one hour on ice (20 mM HEPES, 10 mM MgCl₂, 2 mM EDTA, 5 mM DTT, 0.1 mM Na₂Mo0₄, 1 mM PMSF, 1 mg/ml 4-Aminobenzamidine dihydrochloride, 1 mM NaF, 0.2% Triton X-100, pH 7.5; stock solutions of DTT, PMSF and 4- ABA were added just before use and mixed well). The suspension was shaken vigorously every 15 minutes. To remove nuclei and cell debris, the lysate was centrifuged for 5 minutes at 1000 g. Aliquots of the lysate were used to determine the protein concentration by the method of Bradford (6). The remaining 900 μΐ were centrifuged again under the conditions mentioned above and the cells were resuspended in 3 ml CHCl₃/MeOH (2: 1 v/v). The suspension was shaken vigorously for 1 h at 4°C. After washing with 700 μΐ of a MgCl₂ solution (0.036% in water w/v) for 15 minutes and centrifugation at 300 g for 2 min at room temperature to separate the aqueous phase from the organic phase, the lower chloroform phase was collected and evaporated to dryness under a nitrogen stream.

For mild alkaline hydrolysis, 400 μΐ of CHCl₃/MeOH/H₂0 (16/16/5 v/v/v) were added to the dry lipid extracts and shaken vigorously. After addition of 400 μΐ 0.2 M NaOH in MeOH, the samples were incubated at room temperature for 45 min. Following addition of 400 μΐ 0.5 M EDTA and 150 μΐ CH₃COOH and vigorous shaking, 1 ml CHC1₃ was added to extract the lipids. Extracts were shaken for 5 minutes and centrifuged for 3 minutes at 300 g for phase separation. The chloroform phase was transferred to a new vial and evaporated to dryness under a nitrogen stream. Mass spectrometric analysis of sphingolipids was performed by H. Kofeler (Core Facility for Mass Spectrometry/ Lipidomics, Center for Medical Research, Medical University of Graz) as follows. Dried lipid extracts were resuspended in 100 μΐ CHCi₃/MeOH (1: 1 v/v) containing 100 pmol Cer 12:0, Cer 25:0 and SM 12:0 each as internal standards.

Chromatographic separation of lipids was performed by an Accela HPLC (Thermo

Scientific) on a Thermo Hypersil GOLD C18, 100 x 1 mm, 1.9 μιη column. Solvent A was a water solution of 1 %ammonium acetate (v/v) and 0.1% formic acid (v/v) and solvent B was acetonitrile/2-propanol (5:2, v/v) supplemented with 1% ammonium acetate (v/v) and 0.1% formic acid (v/v), respectively. The gradient was run from 35% to 70% B for 4 min, then to 100% B in additional 16 min with subsequent hold at 100% for 10 min. The flow rate was 250 μΐ/min. Sphingolipid species were determined by a TSQ Quantum Ultra (Thermo Scientific) triple quadrupole instrument in positive ESI mode. The spray voltage was set to 5000 V and capillary voltage to 35 V. SM and Cer species were detected in positive ionization by precursor ion scan on m/z 184 at 35 eV and on m/z 264 at 30 eV, respectively, as described previously (8;9). Cer and SM peak areas were calculated by QuanBrowser for all lipid species and quantification was done by correlation to internal standards.

B) Murine melanoma cells

The uptake and the toxic effects of different naturally occurring and synthetic phospholipids on the murine melanoma cell line B16 were investigated. For this purpose the oxidized phospholipids PGPC and POVPC and the ether-phospholipids E-PGPC and E-POVPC were used (Fig. 1). The two ether-phospholipids E-PGPC and E-POVPC are structurally very similar to their counterparts PGPC and POVPC, but their hydrophobic fatty acid at the sn-l position is linked to the glycerol backbone via an ether bond.

Cell culture and lipid incubation

Purchased B16 murine melanoma cells were cultured in DMEM supplemented with 2% Fetal Bovine Serum and 200 units/ml penicillin/streptomycin, and were routinely grown at 37°C in humidified C0₂ (5%) atmosphere.

For all experiments, ethanolic solutions of lipids containing the indicated μΜ concentration of lipid were prepared using the ethanol injection method (3). The total amount of ethanol was always kept below 1% (v/v). Incubation medium for all experiments was DMEM without phenol red supplemented with 0.1% FBS to avoid degradation of the oxidized phospholipids by high serum concentrations. Control experiments were carried out in DMEM without Phenol red supplemented with 0.1% FBS and the same amount of EtOH but without the lipid. Example 6: PGPC and POVPC reduce the cell viability of B16 mouse melanoma cells in a time- and concentration-dependent manner

To investigate if the oxidized phospholipids PGPC and POVPC have an effect on the cell viability of mouse melanoma cells, the Vybrant® MTT Cell Viability assay was used according to the manufacturer's protocol with slight modifications. For this purpose, B16 cells were incubated with different concentrations (5-250 μΜ) of the indicated lipids for 2 hours, 14 hours and 24 hours in DMEM supplemented with 0.1% FBS. POPC and PLPC were used as native reference phospholipids, and H₂O₂ and staurosporine as positive controls for the induction of cell death.

All tested lipids induce a time- and concentration-dependent decrease in cell viability, but the extent of cell death is dependent on the lipid used (Fig. 7). Regarding the oxidized phospholipids PGPC and POVPC, a reduction of cell viability of B16 cells is already detectable after 2 hours and prolonging the incubation time to 14 hours or 24 hours increases the loss of cell viability. Independent of the incubation time, PGPC is in all cases a more potent inducer of cell death compared to POVPC. The non-oxidized phospholipid POPC does not affect cell viability at all.

Method:

MTT viability assay

To determine the cytotoxic effects of PGPC and POVPC on murine B16 melanoma cells, the Vybrant® MTT Cell proliferation assay was used according to the manufacturer's protocol with slight modification. This assay involves the conversion of water soluble MTT to insoluble formazan by living cells. The purple formazan formed can be solubilised by the addition of SDS and the optical density is measured at 595 nm; high absorbance values correspond with high viability of the cells. In brief, cells were seeded in DMEM

(supplemented with 2% FBS) in 96-well-plates and allowed to reach 80% confluency. The medium was replaced with the lipid dispersion containing varying concentrations (5-250 μΜ) of the indicated lipid or control substances (1% EtOH (v/v) or 1% DMSO (v/v) as negative controls, 2.5 mM H₂O₂ and 10 μΜ staurosporine as controls for necrosis and apoptosis, respectively) for 2 hours, 14 hours or 24 hours. Following incubation, the lipid containing medium was removed and replaced by 100 μΐ fresh medium (0.1% FBS) and 10 μΐ MTT solution (2.5 mg/ml) in PBS prior to incubation for 4 hours at 37°C. Subsequently 100 μΐ, 10% SDS (w/v) in 0.01% HC1 (v/v) were added and the cells were incubated for another 4 hours under the same conditions. After the incubation period, the mixture was resuspended carefully and the optical density was measured at 595 nm using the Anthos plate reader driven by WinRead 2.3 software. Results were obtained from 2 replicates of at least three independent experiments and values represent means + S.D.

Example 7: Oxidized phospholipids and their ether-analogues induce apoptosis in mouse melanoma cells under low serum conditions

MTT experiments showed that incubation of B16 mouse melanoma cells with different oxidized phospholipids leads to a decrease of cell viability already after two hours (Fig. 7). To investigate if this effect is due to an increase in cell death, and if this is caused by necrosis or apoptosis, the occurrence of phosphatidylserine (PS) at the outer leaflet of the plasma membrane was analyzed, which is a typical sign of apoptosis. For this purpose, stimulated cells were stained with green fluorescent AlexaFluor488® Annexin V, which binds to PS on the outer leaflet of the plasma membrane, and red fluorescent propidium iodide (PI), which does not permeate the membrane and thus not stain intact cells (for details see Material and Methods). Cells stained by red PI or double stained cells were defined as necrotic cells, cells showing only the red Annexin V fluorescence were defined to be apoptotic. Living cells were not stained by any fluorescence dye.

B16 mouse melanoma cells were incubated with 25 μΜ or 50 μΜ lipid (PGPC, POVPC, E- PGPC, E-POVPC and Edelfosine) or control substances (1% EtOH or 1% DMEM as negative controls, 10 mM H₂0₂ or 10 μΜ STS as positive controls for induction of necrosis or apoptosis, respectively) for 6 hours in DMEM without Phenol red containing 0.1% FBS. Subsequently the percentage of apoptotic, necrotic and living cells was analyzed.

At low concentrations (25 μΜ) all tested lipids significantly induce apoptosis in B16 mouse melanoma cells (Fig. 8A, Fig. 8B), shown by exposure of phosphatidylserine at the outer leaflet of the plasma membrane; the amount of necrosis is comparable in stimulated and unstimulated cells. The sensitivity of the cells towards treatment with the lipids is highly dependent on the structure and the concentration of the lipid used. PGPC is the most potent inducer of apoptosis. The oxidized diacyl phospholipids PGPC and POVPC elicit more severe apoptotic effects as compared to their alkylacyl counterparts.

However, raising the lipid concentration to 50 μΜ leads not only to an increase in the amount of apoptotic cells, but also to a significant increase of necrosis. It is noteworthy that Edelfosine which was included in this investigation led to a significantly higher percentage of necrotic cells as compared to the oxPL of formula I, in particular at 50 μΜ.

Method:

Flow cytometric analysis of apoptotic and necrotic cells To investigate if the decrease in cell viability after treatment of B16 cells with different lipid compounds is due to necrosis or apoptosis, the Vybrant® apoptosis assay kit#2 was used according to the manufacturer's instructions with slight modifications. Cells were seeded in 24 well plates and allowed to reach 80% confluency. After washing the cells with DMEM containing 0.1% FBS, cells were incubated with 400 μΐ of the lipid dispersion in incubation media containing 25 μΜ or 50 μΜ lipid (PGPC, POVPC, E-PGPC, E-POVPC, Edelfosine), or 400 μΐ incubation media containing control substances (1% EtOH v/v, 10 mM H₂0₂ for induction of necrosis, 10 μΜ STS as positive control for apoptosis, 1% DMSO) for 6 hours. After the incubation period, the supernatant was collected and the cells were harvested using accutase. Wells were washed twice with DMEM to collect any remaining cells and all fractions were combined. After harvesting of the cells by centrifugation, cells were washed with PBS (containing 2% glucose) and resuspended in 100 μΐ Annexin Binding Buffer. 5 μΐ AlexaFluor®488 Annexin 5 and 5.5 μΐ of an aqueous solution of PI (final concentration 1 mg/ml) were added to each sample and incubated in the dark for 15 minutes at room temperature. Samples were diluted with 400 μΐ PBS containing 2% glucose, gently mixed and kept on ice until analysis. Stained samples were analyzed using a FACS Calibur flow cytometer (BD Bioscience, NJ), measuring the green fluorescence emission at 530 nm and the red fluorescence emission above 575 nm upon excitation at 488 nm. Three cell populations were identified: living cells were unstained; cells stained by PI only or cells double stained by PI and Annexin V were defined to be necrotic; cells showing the green AlexaFluor®488 Annexin V fluorescence only, which binds to phosphatidylserine on the outer leaflet of the plasma membrane, but no PI fluorescence, were defined as apoptotic cells. The percentage of apoptotic and necrotic cells was calculated using WinMDI 2.8 software package. Results were obtained from 2 replicates of at least three independent experiments and values represent means + S.D.

Example 8: Activation of acid sphingomyelinase by PGPC and POVPC

Ceramide mediates the cellular response to various stress stimuli. Specifically, it is a key upstream component in many apoptotic signalling pathways. It can be generated by different pathways including the degradation of sphingomyelin by sphingomyelinases, de novo formation from sphinganine by ceramide synthases and the formation of ceramide from sphingosine in the salvage pathway utilizing sphingosine for reacylation by ceramide synthase.

As described above (see (A), Examples 4 and 5) aSMase activity and ceramide formation were stimulated by oxPL in human melanoma cells. According to this study, acid sphingomyelinase activity is activated by oxPL in murine B16 melanoma cells, too.

Specifically, cells were stimulated with 25 μΜ PGPC or POVPC for different times and the rise in aSMase activity was measured (Figure 9). The enzyme was activated by both PGPC and POVPC within minutes and the efficiency of stimulation was similar in both cases. However, at prolonged incubation time (60 min), aSMase returned to control levels in POVPC - treated cells, whereas the enzyme remained active in PGPC - treated cells. This finding is very unusual compared to other cell lines insofar as aSMase activities in vascular cells (vascular smooth muscle cells and macrophages) returned to control levels after an hour (11; 13).

Method:

Determination of acid sphingomyelinase activity

Murine B16-BL6 melanoma cells were grown on 60 mm Petri dishes to 70-80% confluency in DMEM (supplemented with 2% FBS) over night. Prior to stimulation with oxPL, the cells were washed once with medium containing 0.1% FBS. Cells were incubated with 3 ml 25 μΜ aqueous lipid dispersion or 3 ml medium containing 0.1% EtOH (v/v) as a negative control for 15 min, 30 min or 60 min. Following incubation, cells were rinsed with ice-cold PBS, scraped and harvested by centrifugation (300 g, 10 min, 4°C). Cells were lysed in 50 μΐ lysis buffer (250 mM sodium acetate, 0.2% Triton X-100, pH 5.0) for one hour on ice. The suspension was shaken vigorously every 15 minutes. Cell debris and unlysed cells were removed by centrifugation (1000 g, 5 min, 4°C) and the protein content of the samples was determined according to the method of Bradford (6). Aliquots of cell lysate containing 15 μg of protein were incubated with 2 nmol NBD-sphingomyelin in acid reaction buffer for 30 minutes to determine acid sphingomyelinase activity as previously described (7). Fluorescent NBD-sphingomyelin was separated from formed NBD-ceramide by thin-layer

chromatography on silica gel (mobile phase was CHCi₃:MeOH:H₂0 65:24:4 per vol.). The fluorescent spots were quantified with a charge coupled device camera (Herolab, Vienna) at an excitation wavelength of 365 nm using EasyWin software.The ratio of NBD-ceramide to total NBD lipid (Cer + SM) was determined and data (relative aSMase activity) were expressed as means + S.D. (n > 3).

Example 9: OxPL affect ceramide and sphingomyelin species in B16 mouse melanoma cells

As described above (see (A), Example 5) ceramide was formed in cultured human melanoma cells upon exposure to POVPC. This example provides evidence that both PGPC and POVPC lead to a significant rise in total ceramide content after 6 hours (Fig. 10B). PGPC preferentially stimulated formation of C24:0 as well as C24: l ceramide, whereas POVPC only triggered the formation of C24:0 ceramide (Fig. 10A). However, the oxPL-induced formation of ceramide is not associated with significant changes in apparent sphingomyelin patterns. Total sphingomyelin contents remain constant independent of the stimulus, and no significant changes in the amounts of the individual sphingomyelin species can be detected (Fig. IOC and 10D).

Method:

Identification and quantification of ceramide and sphingomyelin species

Murine B16 melanoma cells were grown on 100 mm Petri dishes to 70-80% confluency in DMEM (supplemented with 10% FBS) over night. Prior to incubation, cells were rinsed once with medium containing 0.1% FBS to remove excess serum and incubated with 4 ml of 25 μΜ lipid dispersion in DMEM without Phenol red (0.1% FBS) or control medium containing 1% EtOH (v/v) for 6 hours. Following incubation, cells were rinsed once with ice cold PBS, scraped into PBS and harvested by centrifugation (300g, 10 min, 4°C). Cells were suspended in 1 ml PBS and 100 μΐ aliquots were taken for measuring sample protein concentration. For this purpose, cells were harvested by centrifugation, resuspended in 0.5 ml buffer (50 mM Tris/HCl, pH 7.4) and lysed by sonication (5 pulses a 10 sec). To remove cell debris, the lysate was centrifuged for 5 min at 1000 g and the protein content was determined according to the method of Bradford (6).

The remaining 900 μΐ cell suspension were centrifuged under the conditions described above and cells were suspended in 3 ml CHCl₃:MeOH (2: 1 v/v). The mixture was shaken for 1 h at 4°C. After addition of 700 μΐ MgCl₂ solution (0.036% in H₂0) for 15 min, the upper aqueous phase was removed and the lower chloroform phase was evaporated to dryness under a nitrogen stream. Mild alkaline hydrolysis and detection of the individual ceramide and sphingomyelin species by HPLC-MS were performed as previously described (see (A), Example 5).

Example 10: POVPC decreases undirected cell movement (chemokinesis)

Cancer cells spread from the initial site of tumour growth into surrounding tissues and the vasculature. Preventing metastasis represents an important therapeutic approach to cancer treatment. OxLDL, which contains significant amounts of PGPC and POVPC, has already been known to induce different responses (proliferation or cell death) in vascular cells depending on concentration, incubation time and extent of particle oxidation (14). Thus, the inventors further investigated the effects of lower (5 μΜ or 10 μΜ) oxPL concentrations on proliferation and migration of murine B16 melanoma cells. Figure 11A shows phase-contrast optical micrographs of melanoma cells migrating across a cell free zone after incubation with 5 μΜ oxPL for O h, 10 h and 30 h. Depending on the oxPL used, cells show a different tendency to migrate into this cell free zone over 48 hours. Cells preincubated with the ether-oxPL show the same migration rate compared with control cells (Figure 11C).

Treatment of B16 cells with PGPC leads to an increased motility leading to complete gap closure within 30 hours, whereas incubation with POVPC significantly inhibits cell migration. Most interestingly, higher concentrations of oxPL (10 μΜ) do not show any effects on cell migration. If in contact with POVPC, the cells would even die. They show cell shrinkage, membrane blebbing and detachment from the plate surface (data not shown).

Method:

Determination of melanoma cell migration using a scratch migration assay

Cells were seeded into 24 well plates covered with culture-inserts (Ibidi,

Planegg/Martinsried, Germany) containing full growth medium. Cell were grown over night and allowed to reach 100% confluency. After cell attachment, the Culture-inserts were removed leaving a cell-free gap of approx. 500 μιη width. Remaining cells were washed with medium five times to reduce the number of floating cells that could reattach to the cell-free zone during further incubation. Cells were incubated with 5 μΜ or 10 μΜ oxPL in DMEM (2% FBS) or 1% (v/v) EtOH in DMEM (2% FBS). Cell migration into the cell-free zone under the influence of oxidized phospholipids was followed over a 48 h time period.

Micrographs of the scratch were taken after the indicated incubation times using an Axiovert 35 inverted microscope equipped with a charge-coupled device camera, driven by

Axio Vision software package (Carl Zeiss Vision GmbH, Germany). The microscopy images were always taken from the identical scratch area within one well. The width of the cell-free zone was measured using ImageJ software (Abramoff et ah, Image Processing with ImageJ, 2004, 36-42) and the migration rate was calculated and expressed as percentage of the initial width of the gap. Results were obtained from replicates of three or more independent experiments and data represent means + S.D. (n > 3).

C) Squamous carcinoma cell lines

The uptake and the toxic effects of different naturally occurring phospholipids on different human squamous carcinoma cell lines in comparison to human keratinocytes were investigated. For this purpose the oxidized phospholipids PGPC and POVPC and the ether- phospholipids E-PGPC and E-POVPC were used (Fig. 1). As skin cancer cells the following cell lines were used: HaCaT (immortalized human keratinocytes), SCC12 and SCC13 (established squamous carcinoma cell lines) (Table 1). Table 1 : Characterization and origin of keratinocytes and squamous skin cancer cell lines

Cell culture and lipid incubation

Purchased HaCaT human keratinocytes were cultured in DMEM (4.5 g/1 glucose, 25 mM HEPES, 4 mM L-glutamine, without sodium pyruvate) supplemented with 10% FCS and 100 units/ml penicillin/streptomycin. SCC12 and SCC13 cells (squamous carcinoma cells) were grown in RPMI-1640 medium (supplemented with 10% FCS, 10 units/ml penicillin/ streptomycin, 4 mM L-glutamine). All cell lines were routinely grown at 37 °C in humidified C02 (5%) atmosphere.

All experiments were carried out in RPMI-1640 medium or DMEM without Phenol red (see above) supplemented with 0.1% FCS to minimize degradation of the oxidized phospholipids by lipases under high serum conditions (Fruhwirth et ah, 2006). For all experiments, aqueous lipids suspensions were prepared using the ethanol injection method (3). The total ethanol concentration was always kept below 1% (v/v). Control experiments were performed using incubation medium containing the same amount of ethanol without the lipids.

Example 11 : Effects of oxPL on morphology/integrity of skin cancer cell lines

As described above (see (A)), oxidized phospholipids preferentially induce apoptosis in human melanoma cell lines but not in melanocytes. The same toxic effects of PGPC and POVPC were found in murine B16 melanoma cells (see (B)). It is now demonstrated that oxPL can induce apoptotic cell death also in squamous cell carcinoma cell lines.

Specifically, the toxic lipid effects on squamous carcinoma cell lines and non-tumorigenic HaCaT keratinocytes are presented, which were determined using the following protocol. Cells were incubated with different lipid concentrations for 24 h. Microscopic images were taken after the indicated times to document morphological changes induced by oxPL. Figure 12 summarizes the effects of oxPL on the morphology of HaCaT cells (Figure 12A), SCC12 (Figure 12B) and SCC13 carcinoma cells (Figure 12C).

The observed morphological effects are highly dependent on cell type, lipid structure and lipid concentration. All carcinoma cell lines are highly sensitive towards the treatment with oxPL. At high lipid concentrations, cells are completely lysed. In contrast, HaCaT keratinocytes are only morphologically influenced by incubation with 200 μΜ oxPL.

Method:

Effects of oxPL on cell morphology

75.000 cells were seeded into 24-well plates containing full growth medium and allowed to grow over night. Cells were washed twice with serum-free medium (SFM) to remove floating cells, followed by incubation with different oxPL concentrations. For this purpose, aqueous suspensions containing the indicated concentrations of PGPC or POVPC in medium (0.1% FBS) were prepared as described above. Control cells were incubated with 1% (v/v) EtOH (negative control) or H₂0₂ (necrosis control) in the same medium. Cells were observed with an Axiovert 35 inverted microscope after 6h, 12h and 24h.

Example 12: Effects of oxidized phospholipids on cell viability

Figure 13 shows the oxPL effects on cell viability which were measured using the photometric MTT assay. All lipids under investigation show a concentration-dependent effect of oxPL on the viability of HaCaT cells after 2 hours incubation (Figure 13A). This is surprising insofar as the same cell line is not susceptible to lipid-induced apoptosis (see FACS results, Figure 14). In contrast, the effect of oxPL on SCC13 cell viability was less pronounced (Figure 13B). POPC, which was used as a natural reference phospholipid, did not affect cell viability in any of the cell lines.

Method:

MTT viability assay

The cytotoxic effects of PGPC, POVPC, E-PGPC, and E-POVPC on HaCaT keratinocytes and SCC13 cells were determined using the Vybrant® MTT Cell proliferation assay according to the manufacturer's protocol with slight modifications. The assay is based on the formation of insoluble formazan from water soluble MTT by living cells, and the subsequent solubilisation of the purple formazan crystals by the addition of SDS. Cells were seeded in 96-well-plates using fully supplemented growth medium and allowed to reach 80% confluency. The medium was replaced by fresh medium containing 0.1% FCS and ethanolic solutions of the lipids (concentration range: 5-250 μΜ) or medium containing 1% (v/v) EtOH or DMSO (controls). 2.5 mM H₂0₂ or 10 μΜ staurosporine were added to the medium as positive controls for necrosis or apoptosis, respectively. After incubation at 37°C for 2 hours, the incubation medium was replaced by 100 μΐ fresh medium (0.1% FCS) and 10 μΐ_^ MTT solution (2.5 mg/ml in PBS) and incubated for another 4 hours. Following the addition of 100 μΐ, SDS (10% (w/v) in 0.01% HC1 (v/v)) and cell lysis for 4 hours, the heterogeneous mixture was resuspended carefully and the optical density was measured at 595 nm using the Anthos plate reader driven by WinRead 2.3 software. The decrease in the optical density was determined as a measure for the decrease in cell viability due to a loss of ER and

mitochondrial functions. Results represent means + S.D. of two replicates from three or more independent experiments (n >3).

Example 13: Effects of oxidized phospholipids on cell death

To determine the oxPL capacity of inducing cell death, both cell lines were exposed to 25 μΜ or 50 μΜ oxPL for 6 hours. Apoptotic and necrotic cell populations were identified and analysed by FACS as described below. Staurosporine and hydrogen peroxide were used as control agents to induce apoptosis and necrosis, respectively. 50 μΜ PGPC, POVPC and the respective alkyl ether analogues efficiently induced apoptosis in SCC13 cells, whereas HaCaT cells were almost unaffected (Figure 14). In contrast, the synthetic compound Edelfosine preferentially induced necrosis already at low concentrations (25 μΜ) and higher amounts of this lipid led to complete lysis of the cells.

Method:

Flow cytometric analysis of apoptotic and necrotic cell death

Cells were incubated with 400 μΐ incubation medium containing different oxPL

concentrations (25 μΜ or 50 μΜ) for 6 h or 24 h. Control cells were incubated with medium containing 1% (v/v) EtOH (negative control), 30 mM H₂0₂ (induces necrosis) or 20 μΜ staurosporine (apoptosis inducer). Following incubation, the supernatant was collected and cells were treated with Accutase at 37 °C for 2-3 min to detach the cells from the plate surface. Cell monolayers were washed twice to collect any detached cells and all fractions were combined. After centrifugation, cells were washed once with ice-cold PBS containing 2% glucose (w/v) and resuspended in 100 μΐ Annexin Binding Buffer. For staining, 5 μΐ AlexaFluor®488 Annexin V and 5.5 μΐ propidium iodide (final concentration 1 mg/ml) were added and the mixture was incubated in the dark at RT for 15 minutes. Samples were diluted with 400 μΐ PBS (2% glucose) and FACS analysis of stained cells was performed using a FACS Calibur flow cytometer (BD Bioscience, Heidelberg, Germany). Cells were identified in the side scatter and forward scatter with linear scale. The fluorescence signals were shown and analysed in logarithmic scale. Green (Annexin V) and red (PI) fluorescence emission were measured at 530 nm and 575 nm, respectively, upon excitation at 488 nm.

Three cell populations were identified: Intact cells were unstained, apoptotic cells were stained with green AlexaFluor ®488 Annexin V only, and necrotic cells were either stained by PI only or double stained by PI and Annexin V. The percentage of apoptotic and necrotic cells was calculated using WinMDI 2.8 software package. Results were obtained from three or more independent experiments and values represent means + S.D. (n > 3).

Example 14: OxPL - induced formation of distinct sphingomyelin and ceramide species

Incubation of SCC13 cells with 50 μΜ oxPL for 6 hours is associated with induction of apoptosis. In previous studies it was found that oxPL-induced apoptosis was mediated by an increase in ceramide production in vascular smooth muscle cells (11). As described above (see (A) and (B)), this rise in ceramide was also found in cultured human melanoma cells and murine B16 melanoma cells. The second messenger ceramide propagates apoptotic signalling and can be formed via several pathways, including de novo synthesis, degradation of sphingomyelin and reutilization of sphingosine (salvage pathway). To find out, if apoptosis in SCC13 cells was associated with a rise in cellular ceramide concentrations, cells were stimulated with 50 μΜ PGPC or POVPC for 6 hours and total ceramide was determined as described below.

PGPC does not change ceramide and sphingomyelin levels in SCC13 cells significantly. In contrast, incubation with POVPC leads to a significant rise in total ceramide and also in total sphingomyelin after 6 hours stimulation (Figure 15B).

Method:

Determination of acid sphingomyelinase activity

Cultured SCC13 cells (60 mm Petri dishes, full growth medium, 80% confluency) were washed twice with SFM to remove excess FCS. Subsequently, cells were incubated with 2 ml of 50 μΜ aqueous oxPL dispersion in SFM at 37°C in a humidified 5% C02 atmosphere for 15 min, 30 min or 60 min. Control cells were incubated with SFM containing 1 vol.% EtOH under the same conditions. Following incubation, cells were washed with 3 ml ice-cold PBS, scraped, harvested by centrifugation (1500 rpm, 10 min, 4°C) and lysed by incubation with acid lysis buffer (250 mM sodium acetate, 0.2% Triton X-100, pH 5.0). The protein content of the samples was determined using the method of Bradford (6). Aliquots of cell lysates containing 20 μg protein were used for determination of aSMase activity using fluorescent NBD-SM as a substrate as described previously (7). Briefly, cell lysates were incubated with 2 nmol fluorescent NBD-SM in 200 μΐ acid reaction buffer (250 mM sodium acetate, 1 mM EDTA, pH 5.0) at 37°C for 30 min, followed by lipid extraction with 300 μΐ CHCl₃:MeOH (2: 1). NBD-SM was separated from the formed NBD-CER by thin-layer chromatography on silica gel (mobile phase was CHCl₃:MeOH:H₂0 65:25:4 v/v/v).

Fluorescent spots were quantified with a CCD camera (Herolab, Vienna) (excitation wavelength 365 nm) using EasyWin software. The ratio of NBD-CER to total NBD-lipid (SM+CER) was calculated and data (rel. aSMase activity) were expressed as means + S.D. Results were obtained from three or more independent experiments (n > 3).

Example 15: Stimulation of aSMase activity in SCC13 cells

OxPL-induced apoptosis in SCC13 cells is in part associated with an increase in total ceramide (Figure 15). This effect may be at least in part due to activation of acid

sphingomyelinase. The same phenomenon was observed in vascular cells (VSMCs and RAW 264.7 macrophage) (11; 13) as well as in cultured human melanoma cells and murine B16 cells (see (A) and (B) above). The detailed effects of 50 μΜ PGPC and POVPC on SCC13 cells are illustrated in Figure 16. PGPC induces a significant rise in aSMase activity already after 15 minutes. This activation is lost after 60 min. Surprisingly, POVPC does not activate aSMase in SCC13 cells (Figure 16), although this oxPL stimulates the formation of ceramide (Figure 15). To date it is not known whether the POVPC -induced change in ceramide levels is due to de novo synthesis or the salvage pathway.

Method:

Identification and quantification of total ceramide and sphingomyelin

Cultured SCC13 cells (100 mm Petri dishes, full growth medium, 80% confluency) were washed twice with SFM to remove excess serum. Subsequently, cells were incubated with 4 ml of a 50 μΜ oxPL dispersion in SFM at 37°C in humidified 5% C0₂ atmosphere for 6 hours. Cells incubated with the same amount of SFM containing 1 vol.% EtOH were used as negative controls. Following incubation, cells were washed with ice-cold PBS, scraped into PBS and harvested by centrifugation (1500 rpm, 5 min, 4°C). Cells were resuspended in 1 ml PBS and 100 μΐ aliquots were taken for the determination of sample protein

concentrations by the method of Bradford (6).

The remaining 900 μΐ cell suspension was centrifuged under the conditions described above. Cells were resuspended in 3 ml CHCl₃/MeOH 2: 1 (v/v) and internal standard (5 μg CER 17:0 dissolved in MeOH) was added. The suspension was shaken vigorously at 4°C for 1 hour. The organic phase was washed with 700 μΐ of MgCl₂ solution (0.036% in water w/v) for 15 minutes and centrifuged to facilitate phase separation (300 g, 2 min, RT). The lower chloroform phase was collected and evaporated to dryness under a nitrogen stream.

For mild alkaline hydrolysis, 400 μΐ of CHCl₃/MeOH/H₂0 (16/16/5 per vol.) were added to the solvent-free lipid extracts and the solution was shaken vigorously. After addition of 400 μΐ 0.2 M NaOH in MeOH, the samples were incubated at RT for 45 minutes. Following addition of 400 μΐ 0.5 M EDTA and 150 μΐ CH₃COOH and vigorous shaking, 1 ml CHC1₃ was added to extract the lipids. Extracts were shaken for 5 minutes and centrifuged for 3 minutes at 300 g to facilitate phase separation. The chloroform phase was transferred to a new vial and the solvent was removed under a nitrogen stream.

Evaporated lipid extracts were resuspended in 1 ml CHCl₃:MeOH (2: 1, v/v) and diluted 1:5 with isopropanol. The AQUITY-UPLC system (Waters, Manchester, UK) equipped with a BEH-C18-column, 2,1x150 mm, 1,7 μιη (Waters) was used. For chromatographic separation a binary gradient was applied. Solvent A consisted of H₂0/MeOH (1: 1, v/v), solvent B was 2-propanol. Both solvents contained phosphoric acid (8 μΜ), ammonium acetate (10 mM) and formic acid (0.1 vol%). The column compartment was kept at 50°C. A SYNAPT™ Gl qTOF HD mass spectrometer (Waters) equipped with an ESI source was used for analysis. LeucineEnkephaline [MH+] (m/z 556.2771) was used as reference substance in the lock-spray. Data acquisition was done by the MassLynx 4.1 software (Waters), for lipid analysis the "Lipid Data Analyser" software was used (Hartler et ah, Bioinformatics 27, 2011, 572-577). Results were obtained from 5 independent experiments and data represent means +/- S.D. (n = 5).

Example 16: Influence of oxPL on migration of HaCaT and SCC13 cells

Cancer cells spread from the initial site of tumour growth into the surrounding tissue and eventually form metastatic tumours far distant from the primary lesion. Prevention of this spread represents an ultimate therapeutic approach in cancer treatment. OxLDL, which contains significant amounts of PGPC and POVPC, is known to induce different responses (proliferation or cell death) in cells depending on concentration, incubation time and extent of particle oxidation (14). The inventors found that B 16 mouse melanoma cells under the influence of oxPL showed different tendencies to migrate into a cell free zone (see (B)). PGPC stimulated migration of B16 cells whereas POVPC significantly inhibited migration. In this example, the effects of 5 μΜ and 10 μΜ oxPL on proliferation and migration of HaCaT keratinocytes and SCC13 carcinoma cells were determined. Figure 17 summarizes the results. Typically, cells were incubated with oxPL and their migration into a cell-free zone was determined over 9 hours until closure of the cell-free zone was detectable. 5 μΜ oxPL does not significantly change migration of both cell types. A slightly faster migration of keratinocytes was observed when the cells were exposed to the ether phospholipids. Much faster migration of HaCaT keratinocytes is seen after pretreatment with 10 μΜ E-PGPC. In contrast, migration of SCC13 cells is not altered by oxPL.

Method:

Determination of cell migration using a scratch migration assay

Cells were seeded into 24 well plates covered with culture-inserts (Ibidi,

Planegg/Martiensried, Germany) containing full growth medium over night and allowed to reach 100% confluency. Culture-inserts were carefully removed, leaving a cell-free gap of about 500 μιη width. Subsequently, remaining cells were rinsed with medium several times to reduce the number of floating cells that could reattach to the cell-free zone during further incubation. Following incubation with different oxPL concentrations in DMEM or

RPMI-1640 medium (2% FCS), or 1% (v/v) EtOH in the same media (negative controls), cell migration into the cell-free zone was observed. Micrographs of the scratch were taken after the indicated incubation times using an Axiovert 35 inverted microscope equipped with a CCD camera, driven by Axio Vision software package (Carl Zeiss Vision GmbH,

Germany). All images were taken from the identical scratch areas within one well. The width of the cell-free zone was measured using ImageJ software (Abramoff et ah, 2004). The migration rate was calculated and expressed as % of the initial width of the gap. Results were obtained from replicates of three or more independent experiments and data represent means + S.D. (n > 3).

Reference List:

1. Moumtzi, A., Trenker, M., Flicker, K., Zenzmaier, E., Saf, R., and Hermetter, A. (2007) J. Lipid Res. 48, 565-582

2. Hermetter, A., Stutz, H., Franzmair, R., and Paltauf, F. (1989) Chemistry and Physics of Lipids 50, 57-62

3. Batzri, S. and Korn, E. D. (1973) Biochim. Biophys. Acta 298, 1015-1019

4. Fruhwirth, G. O., Moumtzi, A., Loidl, A., Ingolic, E., and Hermetter, A. (2006) Biochim. Biophys. Acta 1761, 1060-1069

5. Vaskovsky, V. E. and Kostetsky, E. Y. (1968) J. Lipid Res. 9, 396

6. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254

7. Loidl, A., Claus, R., Deigner, H. P., and Hermetter, A. (2002) J. Lipid Res. 43, 815-823

8. Bielawski, J., Pierce, J. S., Snider, J., Rembiesa, B., Szulc, Z. M., and Bielawska, A. (2009) Methods Mol. Biol. 579, 443-467

9. Brugger, B., Erben, G., Sandhoff, R., Wieland, F. T., and Lehmann, W. D. (1997) Proc. Natl. Acad. Sci. U. S. A 94, 2339-2344

10. Park, C. H., Kim, M. R., Han, J. M., Jeong, T. S., and Sok, D. E. (2009) Lipids 44, 425- 435

11. Loidl, A., Sevcsik, E., Riesenhuber, G., Deigner, H. P., and Hermetter, A. (2003) /. Biol. Chem. 278, 32921-32928

12. Stemmer, U., Ramprecht, C, Zenzmaier, E., Stojcic, B., Rechberger, G., Kollroser, M., Hermetter, A., (2012) Biochim. Biophys. Acta 1821, 706-718

13. Stemmer, U., Dunai, Z. A.., Koller, D., Purstinger, G., Zenzmeier, E., Deigner, H. P., Aflaki, E., Kratky, D., Hermetter, A., (2012) Lipids Health Dis 11, 110 14. Han, C. Y., Pak, Y. K, (1999) Exp. Mol. Med. 31, 165-173

Claims

Claims:

1. A phospholipid compound of formula I

wherein R¹ is selected from the group consisting of:

-CO(CH₂)_pCHO and

-CO(CH₂)_pCOOH, wherein p = 1-7,

and R is selected from the group consisting of:

-CO(CH₂)_nCH₃ and

-(CH₂)_mCH₃, wherein n = 8-16 and m = 8-20,

for use in the treatment of skin cancer and precancerous skin lesions.

2. A phospholipid compound for use according to claim 1, wherein R is - CO(CH₂)_nCH₃.

3. A phospholipid compound for use according to claim 2, wherein R¹ is - CO(CH₂)_pCHO, p is 3 and n is 14.

4. A phospholipid compound for use according to claim 2, wherein R¹ is - CO(CH₂)_pCOOH, p is 3 and n is 14.

5. A phospholipid compound for use according to claim 1, wherein R is -(CH₂)_mCH .

6. A phospholipid compound for use according to claim 5, wherein R¹ is - CO(CH₂)_pCHO, p is 3 and m is 15.

7. A phospholipid compound for use according to claim 5, wherein R¹ is - CO(CH₂)_pCOOH, p is 3 and m is 15.

8. A phospholipid compound for use according to any of claims 1 to 7, wherein the skin cancer is selected from primary melanomas and metastatic melanoma.

9. A phospholipid compound for use according to claim 8, wherein the primary melanomas are selected from the group consisting of lentigo maligna, lentigo maligna melanoma and primary melanomas which are not accessible to surgery.

10. A phospholipid compound for use according to any of claims 1 to 7, wherein the skin cancer is a non-melanoma skin cancer.

11. A phospholipid compound for use according to claim 10, wherein the non-melanoma skin cancer is a basal-cell carcinoma or a squamous cell carcinoma.

12. A phospholipid compound for use according to any of claims 1 to 7, wherein the precancerous skin lesion is actinic keratosis.

13. A phospholipid compound for use according to any of claims 1 to 7, wherein the phospholipid compound is used for the topical treatment of skin cancer and precancerous skin lesions.

14. A pharmaceutical composition for use in the treatment of skin cancer and

precancerous skin lesions, comprising a phospholipid compound of formula I

wherein R is selected from the group consisting of:

-CO(CH₂)_pCHO and

-CO(CH₂)_pCOOH, wherein p = 1-7,

and R is selected from the group consisting of:

-CO(CH₂)_nCH₃ and

-(CH₂)_mCH₃, wherein n = 8-16 and m = 8-20, and a pharmaceutically acceptable carrier therefor.

15. The pharmaceutical composition according to claim 14 for topical application.