Patent Application
20160067211
The present invention relates to a pharmaceutical composition for the prophylaxis and treatment of a melanoma or a precursor thereof, as well as a skin or mucosa metastasis, and uses and methods associated therewith.
The malignant melanoma is a highly malignant tumor of the pigment cells, the so-called melanocytes. It tends to spread metastases via the lymphatic and blood circulation at an early stage and is the most frequently lethal skin disease with a worldwide rapidly growing number of new cases.
Mostly the melanoma develops from a precursor. This group of cells already contains melanoma cells, which however are locally confined at the upper tissue layer. It is also referred to a melanoma in situ. One of the precursors of the malignant melanoma is defined as Lentigo maligna.
In the literature a distinction is drawn between at least five different subtypes of the malignant melanoma. The most frequent form is the superficial spreading melanoma (SSM). The most aggressive form of the malignant melanoma with the most unfavorable prognosis is the nodular malignant melanoma (NMM). Further subtypes are the lentigo maligna melanoma (LMM), the acral lentiginous melanoma (ALM) and the amenalotic melanoma (AMM).
Melanomas can metastasize into different organs, preferred target organs such as for other tumors do not exist. Very often metastases can be found in the liver, in the skin, in the lung, in the skeleton and in the brain.
Skin metastasis, also referred to as secondary skin cancer or metastatic skin cancer, is defined as a lymphogenic or hematogenic sealing of primary skin malignomas or tumors of other organs in the skin. As causal primary tumors in particular the following tumors come into questions in descending order: The malignant melanoma, mamma carcinoma, gestric carcinoma, uterus carcinoma, bronchial carcinoma, rectal carcinoma or renal carcinoma. They can appear anywhere on the body surface, preferably on the abdominal wall, the trunk and the capillitium.
The most important form of therapy of the melanoma is still the surgical removal of the primary tumor and the excision of the metastases. Only the complete removal of the primary tumor at an early stage can result in healing. At later stages, where the tumor has already formed metastases in the skin, lymph nodes and internal organs, the chance for cure is low. This is where a good number of therapy alternatives are applied and tested which usually only provide a temporary recovery, however in most cases do not have a prospect of cure. These include chemotherapy with DTIC or fotemustin, a vaccination therapy with antigen presenting cells, surgical interventions to reduce the tumor mass, or a radiation therapy. More recent approaches of therapy are based on the blockade of molecular processes in the signal transduction of the cell. Here, for example, combinations of a traditional chemotherapeutic agent with b-RAF Kinase inhibitors such as sorafenib are used.
A very common form of the treatment of skin metastases is also the intralesional injection with Interleukin-2 (IL-2).
The surgical removal of the melanoma, its precursors or metastases is not always possible. In addition, the intervention is burdensome for the patient, painful after the operation, time-consuming and logistically complicated, in particular when there are long distances to the clinic or a critical general condition of the patient. Furthermore, the intervention is associated with scar formation and generally bears the risk of wound infections.
The intralesional injection of IL-2 also has significant disadvantages. It has to be carried out experimentally under study conditions over a longer period of time, three times a week in a skin center. Besides the high expenditure and the costs associated therewith this option of treatment is very often accompanied by severe and in parts systemic side effects. The application as such is painful and bears the risk of skin infections. Furthermore the IL-2 injection is only appropriate for skin metastases with a diameter of <5 mm and has only moderate response rates.
Because of the strongly increasing number of new cases and the therapeutic approaches which are so far not satisfying there is a strong need for new treatment options.
Against this background it is an object of the present invention to provide a new pharmaceutical composition, by means of which a melanoma and a melanoma precursor as well as a skin and mucosa metastasis of any kind of primary tumor can be treated or prevented, respectively.
This object is met by the provision of a pharmaceutical composition which comprises as an active agent a prenylflavonoid, preferably prenylnaringenin (PN).
Prenylflavonoids belong to the group of flavonoids, a large group of lower molecular weight polyphenolic compounds. Flavonoids can be found in plants and consist of flavones, flavonoles, flavonones, flavan-3-oles and anthocyanines. The secondary plant metabolites play a role in the defense of the plant against micro-organisms or fungi and the protection against oxidative stress.
An important representative of the prenylflavonoids is the prenylnaringenin (PN).
It is understood that the pharmaceutical composition according to the invention can comprise a pharmaceutically acceptable formulation. Pharmaceutically acceptable formulations are well known in the state of the art. Exemplarily, it is referred to the essay of Kibbe A. (2000), Handbook of Pharmaceutical Excipients, 3rd edition, American Pharmaceutical Association and Pharmaceutical Press. The pharmaceutical composition according to the invention may also comprise additives. They include any compound or composition which is beneficial for the use according to the invention, which encompass salts, binding agents, solvents, dispersing agents and additional compounds which are usually applied in the formulation of medicinal products.
The object underlying the invention is herewith completely met.
According to an embodiment of the invention the pharmaceutical composition comprises 6-Prenylnaringenin (6-PN) and/or 8-Prenylnaringenin (8-PN).
6-PN and 8-PN are so-called prenylflavonoids. The can be found in low concentrations, e.g. in hops and in beer. The structure of this class of flavonoids is derived from 2-phenylchrom-4-on. 6-PN and 8-PN are isomers. The difference is in the position of the prenyl group consisting of five carbon atoms. 6-PN and 8-PN are products of a ring closure reaction of a common precursor molecule, desmethylxanthohumol, a polyphenol, which is based on the structure of 1,3-diphenyl-2-propene-1-one. Both molecules exist as enantiomeres (R, S).
8-PN has a strong binding affinity for estrogen receptors of the rat uterus and has been identified as a potent phytoestrogen due to a characteristic spacing of the hydroxyl groups, which imitate beta-estradiol; cf. Milligan et al. (1999), Identification of potent phytoestrogen in hops (Humulus lupulus L.) and beer, J. Clin. Endocrinol. Metab. 84, 2249-2252.
Apart from the antioxidative properties some flavonoids also have anticancer properties; cf. Hodek et al. (2002), Flavonoids-potent and versatile biologically active compounds interacting with cytochromes p. 450, Chem. Biol. Interact. 139, 1-21; Son et al. (2007), Pomiferin, histone deacetylase inhibitor isolated from fruits of Maclura pomifera, Bioorg. Med. Chem. Lett., Vol. 17(17), 4753-4755; Cidade et al. (2001), Artelastocarpin and carpelastofuran, two new flavones, and cytotoxicities of prenylflavonoids from Artocarpus elasticus against three cancer cell lines, Planta. Med. Vol. 67(9), 867-870; Kuete et al. (2011), Cytotoxicity and mode of action of four naturally occurring flavonoids from the genus Dorstenia: gancaonin Q, 4-hydroxylonchocarpin, 6-prenylapigenin, and 6,8-diprenyleriodictyol, Planta. Med. Vol. 77(18), 194-1989 (abstract).
8-PN inhibits the angiogenesis, induced by the basic fibroblast growth factor and the vascular endothelial growth factor in a three-dimensional collagen gel in vitro and in chorioallantois membrane assays in vivo; cf. Pepper et al. (2004), 8-Prenylnaringenin, A novel phytoestogen, inhibits angiogenesis in vitro and in vivo, J. Cell Physiol. 199, 98-107.
8-PN imitates the effects of 17β-Estradiol on the breast cancer cells MCF-7; cf. Rong et al. (2001), 8-Prenylnaringenin, the phytoestrogen in hops and beer, upregulates the function of the e-cadherin-/catenin complex in human mammary carcinoma cells, Eur. J. Cell Biol. 80, 5 580-585.
8-PN further induces the apoptosis in MCF7-cells and in a leukemia blast; cf. Brunelli et al. (2007), 8-Prenylnaringenin, inhibits estrogen receptor-α mediated cell growth and induces apoptosis in MCF-7 breast cancer cells, J. Steroid. Biochem. Mol. Biol. 107, 140-148, and Diller et al. (2007), Ability of prenylflavanones present in hops to induce apoptosis in a human Burkitt lymphoma cell line, Planta Med. 73, 755-761.
Recently also described was the inhibition of the P-glycoprotein, the transporter protein associated with multi resistency, and the inhibition of MRP1 by 8-PN; cf. Wesolowska et al. (2010), 8-Prenylnaringenin is an inhibitor of multidrug resistance-associated transporters, P-glycoprotein and MRP1, Eur. J. Pharmacol. 644, 32-40.
In addition, 8-PN directly inhibits the activation of the PI (3) K/Akt-signal path in MCF-7 cells in vitro; Brunelli et al. (2009), 8-Prenylnaringenin inhibits epidermal growth factor-induced MCF-7 breast cancer cell proliferation by targeting phosphatidylinositol-3-OH kinase activity, J. Steroid. Biochem. Mol. Biol. 113, 163-170.
6-PN and 8-PN show antiproliferative effects on the human prostate cancer cell lines PC-3 and DU145 in vitro; Delmulle et al. (2006), Anti-proliferative properties of prenylated flavonoids from hops (Humulus lupulus L.) in human prostate cancer cell lines, Phytomedicine 13, 732-734. This happens in the absence of a Caspase-3 activation and typical apoptotic morphological characteristics; Delmulle et al. (2008), Treatment of PC-3 and DU145 prostate cancer cells by prenylflavonoids from hop (Humulus lupulus L.) induces a caspase-independent form of cell death, Phytother. Res. 22, 197-203.
The suitability of 6-PN and 8-PN for the prophylaxis and treatment of a melanoma or a precursor thereof as well as a skin or mucosa metastasis of any primary tumor is so far not described in the state of the art or rendered obvious. This is also because the exact cellular mechanisms of 6-PN and 8-PN are not known so far.
The inventors were now able to demonstrate for the first time in silico and in vitro that 6-PN and 8-PN are distinct potent inhibitors of histone deacetylase (HDACs) of the classes I, II and IV and induce a hyperacetylation of the histone protein H3. This could be shown by the inventors using the example of different tumor cell lines, in particular by means of both of the melanoma cell lines SK-MEL-28 and BLM.
In addition, the inventors were able to demonstrate in a large number of tumor cell lines that 6-PN and 8-PN show strong apoptosis independent antiproliferative properties in vitro and low toxicity in vivo. In human epidermal skin reconstructions both PNs show a strong inhibited cell proliferation and invasion of the tumor cells.
Furthermore, the inventors were able to observe the induction of autophagy in tumor cells after the treatment with 6-PN and 8-PN.
Taken together, the data generated by the invention show that 6-PN and 8-PN are potent phytotherapeutic epigenetic active agents for the melanoma prevention, melanoma therapy, treatment and prophylaxis of skin and mucosa metastasis of any primary tumor.
According to an embodiment of the invention the pharmaceutical composition is configured for the prophylaxis and/or treatment of a melanoma or a melanoma precursor of the skin.
This measure has the advantage that the medicinal product is provided for the treatment of the most prevalent melanoma form, namely the cutaneous melanoma and its precursors. So far satisfying therapeutic approaches against such clinical pictures do not exist in the state of the art.
According to another embodiment of the invention the pharmaceutical composition is configured for the prophylaxis and/or treatment of melanoma skin and/or melanoma mucosa metastasis.
Also with this measure the treatment of such metastasis is addressed in a preferred manner for which so far no sufficient concepts of treatment do exist.
According to an embodiment of the invention the pharmaceutical composition is configured for a topical application, further preferably for a topical application onto the skin.
This measure has the advantage that for the first time an effective topical therapy of the melanoma, its precursors or of metastases of any primary tumor is possible, which can be carried out by the patient itself. For the patient it is far less burdensome, both physically as well as mentally, as current forms of therapy, for example the injection of the lesions with IL-2. Compared to surgical procedures and the IL-2 injection also the cost expenditure of such a topical therapy is significantly reduced. Also the risk of infections is strongly diminished. The topical configuration of the composition according to the invention furthermore allows the treatment of a larger skin area. According to findings of the inventors a better anti-tumor effect and a higher response rate can be expected in contrast to the IL-2 injection. In addition, the topical application allows the treatment of occult skin micro-metastases. As a consequence, a recurrence can be counteracted which is only partially or not at all the case in a surgical removal and an IL-2 injection. In addition, the topical application allows an effective melanoma prophylaxis, the therapy of melanoma precursors or, after the excision of a primary melanoma, the reduction of the risk of the primary melanoma formation or a melanoma recurrence, respectively.
Therefore, according to another embodiment of the pharmaceutical composition is provided in an application form which allows a topical application and is, for this reason, preferably selected from the group consisting of: ointment, cream, lotion, gel, paste, transdermal therapeutic system, foam, powder.
The active agents 6-PN and/or 8-PN can be provided in encapsulated form, for example in liposomes.
The composition according to the invention can be configured as a mono-preparation, where 6-PN and/or 8-PN are provided as (the) only active agent(s). According to a further embodiment of the invention the pharmaceutical composition comprises an additional active agent effective against melanomas and/or melanoma precursors and/or skin and/or mucosa metastases of any primary tumor.
This measure may take advantage of synergistic effects resulting from the cooperation of 6-PN and/or 8-PN and the additional active agent. This may also result in a simplification of the application by the patient who may apply only the pharmaceutical composition of the invention instead of two preparations.
In an embodiment of the invention the additional active agent is a necrosis inhibitor.
This measure has the advantage that necrotic processes and inflamations associated therewith are inhibited and any side effects of the composition according to the invention are further reduced.
According to another embodiment of the invention it is preferred if the necrosis inhibitor is IM-54.
By this measure such a necrosis inhibitor is used which is especially suited according to the findings of the inventors.
Another subject matter of the present invention relates to the use of prenylnaringenin, preferably 6- and/or 8-Prenylnaringenin, as an epigenetically active medicinal agent, further preferably for the prophylaxis and/or treatment of a melanoma and/or a melanoma precursor and/or a skin and/or mucosa metastasis of any primary tumor.
Another subject matter of the present invention relates to the use of prenylnaringenin, preferably 6- and/or 8-prenylnaringenin, as a histon deacetylase inhibitor (HDACi).
Another subject matter of the present invention is a method for the prophylaxis and/or treatment of a melanoma and/or a melanoma precursor and/or a skin and/or mucosa metastasis of any primary tumor in a living being, preferably a human being, comprising the following steps: (1) Providing the pharmaceutical composition according to the invention, and (2) application of the pharmaceutical composition onto the skin of the living being, preferably the melanoma and/or the melanoma precursor and/or the skin and/or mucosa metastasis.
The properties, features and advantages of the pharmaceutical composition according to the invention apply likewise to the uses according to the invention and the method according to the invention.
It goes without saying that the features mentioned before and those that will be explained hereinafter may not only be used in the particularly given combination but also in other combinations or alone without departing from the scope of the present invention.
The invention is now explained in further detail by means of embodiments from which further properties, features and advantages will result. The embodiments shall illustrate the invention, however do not restrict its scope. Reference is made to the enclosed figures:
1.1 Synthesis of 6-prenylnaringenin and 8-prenylnaringenin (PN)
8-Prenylnaringenin and 6-prenylnaringenin were synthesized according to Gester et al. (2001), An efficient synthesis of the potent phytoestrogens 8-prenylnaringenin and 6-(1,1-dimethylallyl)naringenin by europium (III)-catalyzed Claisen rearrangement, Tetrahedron 57, 1015-1018 and Tischer and Metz (2007), Selective C-6 prenylation of flavonoids via europium(III)-catalyzed Claisen rearrangement and cross-metathesis, Advanced Synthesis & Catalysis 349, 147-151, synthetized. The content of both before-mentioned publications is incorporated herein by reference.
The inventors have tested 19 different tumor cell lines in total, including five melanoma cell lines, two liver cancer cell lines, three breast cancer cell lines, three colon cancer cell lines, two prostate cancer cell lines, two lung cancer cell lines and to renal cancer cell lines.
As example in the following metastatic melanoma cell lines SK-MEL-28, LOXIMVI and BLM, the colon cancer cells HT29 and the breast cancer cells MCF-7 are described. They were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS), 1% Penicillin, and 1% Streptomycin. All cell cultures were maintained at 37° C. in a 95% air/5% CO2 atmosphere at 100% humidity.
The docking analyses were performed with human HDAC2, 4, 7, and 8 with 6-PN, 8-PN, and the two reference HDACi compounds suberoyl anilide hydroxamic acid (SAHA) and trichostatin A (TSA).
The determination of the HDACi activity was performed with the HDAC assay kit as described by the manufacturer (Active Motif, La Hulpe, Belgium), using 6-PN and 8-PN at rising concentrations (5 μM, 10 μM, 20 μM, 50 μM and 100 μM).
The human HDAC profiling assay was performed on the basis of the Fluor de Lys™ technology by Scottish Biomedical, Glasgow, United Kingdom. The percentage inhibition values of 100 μM of 6-PN and 100 μM of 8-PN against the human HDAC enzymes HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, and HDAC11 were determined. All assays were performed in 1% DMSO (final concentration).
The following antibodies were used: Anti-Vinculin and Anti-Actin (1:5000, Sigma-Aldrich), Anti-Acetyl-Histon H3 (1:5000, Millipore, Billerica, USA), Anti-Caspase-3 (1:1000), Anti-AKT, Anti-pAKT, Anti-ERK, Anti-pERK, Anti-pE90, Anti-p70S6 Kinase, and Anti-pS6 protein (1:1000), all from Cell Signaling, Frankfurt, Germany), Anti-LC3 (1:5000, Sigma-Aldrich).
The cells were seeded in triplicates in 96-well plates at a density of 2,500 cells per well in 50 μl medium (5×104 cells per milliliter). After 24 hours, the medium was replaced by medium containing 6-PN, 8-PN (dissolved in DMSO), TSA (dissolved in EtOH) or SAHA (dissolved in DMSO) at the concentrations to be tested with or without the addition of the necrosis inhibitor IM-54 at 5 μM (Calbiochem, Darmstadt, Germany). Cells treated with culture medium without or with DMSO served as controls. The assay was started following an incubation of 24 hours. The medium was discarded, each well was washed two times with PBS (without Ca++ and Mg++) and 100 μl of a solution containing 100 mg of 4-methyl umbellipheryl heptanoate per ml PBS was added. Plates were incubated at 37° C. for 1 hour and measured in a Fluoroskan II (Lab Systems, Helsinki, Finland) with a λem of 355 nm and λex of 460 nm. The intensity of fluorescence indicates the number of viable cells in the wells.
The human melanoma cell lines BLM and SK-MEL-28 (2.5×103 cells/well) were seeded in 96 well plates. The cells were treated after 24 hours with different concentrations of 6-PN or 8-PN (20 μM, 50 μM and 100 μM) and monitored by measurements of electrical impedance in 15 minutes intervals for an overall time span of in total 104 hours using the xCELLigencer® SP Systems (Roche Applied Science). The cell index values were calculated using the RTCA software (1.0.0.0805). All curves were normalized at the beginning of the treatment period.
Testing of apoptosis induction was performed with the human Proteom-Profiler-Apoptosis-Antibody-Array-Kit (R & D Systems, Wiesbaden, Germany) as described by the manufacturer after treatment of SK-MEL-28 melanoma cells with 100 μM 6-PN or 100 μM 8-PN for 4 hours.
Organtypic cultures of human skin and melanoma (using BLM and LOXIMVI melanoma cells) were generated as previously described by Meier et al. (2000), Human melanoma progression in skin reconstructs: biological significance of bFGF, Am. J. Pathol. 156, 193-200.
Fertilized eggs of leghorn chickens (Gallus gallus domesticus) were incubated at 38° C. in a temperature-controlled, humidified brooder. The upper most spot of the egg shell and thus indirectly the blastoderm, which is always oriented towards the top part of the egg, was marked on each egg with a permanent marker. For embryo toxicity testing, the eggs were prepared after approximately 48 to 52 hours of incubation (equal to stage 12 to 13), which corresponds to approximately 6 human gestational weeks. Before fenestration, a small hole was pierced into the lateral edge of the eggs and 2 ml of albumen were withdrawn with a syringe from the lower level of the blastoderm. Subsequently, the egg was prepared for fenestration by using a hack saw to generate a rectangular predetermined breaking point on the shell around the previously marked spot (about 5×25 mm in size). The predetermined “window” was opened by removal of the egg shell with bent forceps. At this stage, the embryo is already visible on top of the blastoderm. 100 μM of 6-PN or 8-PN (dissolved in 50% ethanol and 50% PBS, total volume: 500 μl) was applied on the top of the blastoderm (2 embryos per treatment group; the experiment was performed in triplicates; total n=6 per group). As control, the same number of embryos were treated with the same volumes of ethanol and PBS without the addition of 6-PN or 8-PN to exclude possible toxic effects of ethanol. The eggs were then sealed with adhesive tape and replaced into the incubator. The viability of the embryos was verified and documented 24, 48 and 72 hours after the application of the substances.
Statistical analyses for different assays were performed with the One-Way ANOVA Dunnett's Multiple Comparison Test using GraphPad Prism Version 4.00 (GraphPad Software, San Diego, Calif., USA). According to this analysis a value of p<0.01 was defined as statistically significant.
2.1 in Silico Docking Analyses Predict Binding of 6-PN and 8-PN into Human HDAC Enzymes.
To identify potential HDACi activity of 6-PN and 8-PN (
Based on the in silico data 6-PN and 8-PN seem to exhibit inhibitory activity against HDAC enzymes of class I and II comparable to standard HDACi like TSA and SAHA.
To further substantiate the predicted HDACi activity in vitro, 6-PN and 8-PN were screened in a HDACi assay. Employing standardized nuclear extract of HeLa cells as human HDAC enzyme source, 6-PN and 8-PN showed a dose dependent HDAC inhibitory activity (
To prove the in silico and in vitro results and to apportion the inhibitory activity of 6-PN and 8-PN, a profiling analysis with all known human HDAC enzymes of class I, II and IV was performed (
2.3 Hyperacetylation of Histon H3 after 6-PN and 8-PN Treatment in BLM and SK-MEL-8-Melanoma Cells
An incubation of cells with HDACi induces generally a hyperacetylation of histon proteins. Therefore, the acetylation status of the histon protein H3 after treatment with 6-PN or 8-PN was investigated. Corresponding to the HDAC screening results, a direct effect was monitored. Western blot analyses revealed a massive increase of histon complex H3 acetylation in BLM melanoma cells 24 hours after treatment with 100 μM of 6-PN or 8-PN, and in SK-MEL-28 melanoma cells already two hours after the treatment with 100 μM of 6-PN or 8-PN, when compared to DMSO treated control cells (
Due to the new found HDACi activity of 6-PN as well as 8-PN and the observed hyper acetylation of histon protein H3 in human melanoma cells, the antiproliferative effects on cancer cells were tested. The human metastatic melanoma cell lines BLM and SK-MEL-28 were treated once with rising concentrations of 6-PN or 8-PN (20 μM, 50 μM and 100 μM) and monitored continuously for an overall time span of 104 hours using a real time monitoring assay (
In order to substantiate the antiproliferative effects of 6-PN and 8-PN on BLM and SK-MEL-28 melanoma cells, proliferation assays were performed (
To further evaluate the anti cancer properties of 6-PN and 8-PN on metastatic melanoma cells the proliferation assays were repeated with the addition of the necrosis inhibitor IM-54 to analyze the role of necrosis induction upon the treatment with PN. The addition of 5 μM IM-54 decreased cell death rates at higher PN concentrations in both BLM and SK-MEL-28 cell lines by up to 35% (
Next it was asked whether the PNs also influence the proliferation of benign cells. To this end, human fibroblasts (FF) and foreskin melanocytes (HEM1) were treated with the same concentrations of 6-PN and 8-PN, and proliferation was assessed using the proliferation assay as described above. While fibroblast proliferation was hardly affected by the PN treatment (
Taken together, 6-PN and 8-PN effectively reduce cell proliferation in all tested cancer cells (cf. Tab. 1); especially melanoma cells were highly susceptible towards the treatment with the two prenylflavonoids. IM-54 was able to partially block PN-induced cell death in BLM and SK-MEL-28 cells, suggesting that the antiproliferated and cytotoxic cascade initiated by PN eventually might lead to the induction of necrosis in a fraction of the melanoma cells.
To further analyze the type of cell death induced by 6-PN and 8-PN, western blot analyses of SK-MEL-28 melanoma cells were performed 1, 2, 4, 12 and 24 hours after the treatment with 100 μM 6-PN and 8-PN. For both PNs decreased protein levels of caspase-3 (proapoptotic) and Bcl-xl (anti-apoptotic) were detected (not shown). However, no additional band for the cleaved caspase-3 for 6-PN, 8-PN or any of the applied reference HDACi (TSA, SAHA) or the vehicle control (DMSO) in SK-MEL-28 cells was detected (
To further analyze the possibility of apoptosis induction, an extensive apoptosis assay was performed on SK-MEL-28 melanoma cells 4 hours after treatment with 100 μM 6-PN or 8-PN (
Since it has been speculated that the formation of intracytoplasmic vacuoles in pancreatic or breast cancer cells by 6-PN and 8-PN observed in pervious studies might represent an induction of autophagy, this issue was further investigated on a molecular level. BLM and SK-MEL-28 melanoma cells were treated with 100 μM of 6-PN, 8-PN, SAHA or TSA (and with DMSO as control), and the treated cells were lysed for protein extraction after 1 hour, 2 hours, 4 hours, 12 hours and 24 hours. As indicator for the induction of autophagy, the shift from LC3 (LC3-I) to the autophagic, vesicle-associated form (LC3-II), was used. In both melanoma cells after 12-24 hours only 6-PN and 8-PN had clearly induced the shift from LC3-I to LC3-II (
Since 6-PN and 8-PN had shown to exert antiproliferative effects on melanoma cells in vitro, it was asked whether such effects could be visualized in a more sophisticated human epidermal skin reconstruct. In this assay, keratinocytes and melanoma cells are seeded on top of a collagen matrix populated by fibroblasts. After 16 days a keratinizing stratified epithelium with a rudimentary corium is formed. Untreated control BLM cells (depicting 70-80% proliferation, determined by MIB1 immunhistochemistry) had formed a large tumor in the epidermal part of the reconstruct and had invaded the corium (
Together, the antiproliferative effects observed in vitro could be reproduced and extended to an addition inhibition of BLM and LOXIMVI melanoma cell invasion in epidermal skin reconstructs.
2.7 In Vivo Toxicity Studies with 6-PN and 8-PN
Due to the detected cytotoxic effects on cancer cells and of melanocytes in vitro, the determination of in vivo embryotoxicity was of great interest. To this end, 2-day chicken embryos were treated for 24, 48 and 72 hours with 100 μM 6-PN or 100 μM 8-PN to monitor survival as readout for lethal embryotoxicity (
The inventors were able to demonstrate on the basis of 19 different tumor cell lines in an impressive manner that a prenylflavonoid or prenylnaringenin (PN), respectively, in particular 6- and/or 8-prenylnaringenin (6-PN, 8-PN), is suited for the prophylaxis and/or treatment of a melanoma and/or a melanoma precursor and/or a skin and/or mucosa metastasis.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10 2013 104 342.9 | Apr 2013 | DE | national |
This application is a continuation of copending International Patent Application PCT/EP2014/056464 filed on Mar. 31, 2014 and designating the United States of America, which was not published under PCT Article 21(2) in English, and claims priority of Germany Patent Application DE 10 2013 104 342.9 filed on Apr. 29, 2013, which are both incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/EP2014/056464 | Mar 2014 | US |
| Child | 14925859 | US |
