PORCINE IMMUNE MODULATION MODEL

Abstract

A method of modulating an immune response. The method includes administering a treatment comprising delta 5-androsten-3B, 17B-diol, delta 5-androsten-3B, 7B, 17B-triol, or both.

Claims

1. A method of modulating an immune response, the method comprising: administering a treatment comprising delta 5-androsten-3B, 17B-diol, delta 5-androsten-3B, 7B, 17B-triol, or both.

2. The method of claim 1, wherein the treatment further comprises: one or more of estradiol, testosterone, dihydrotestosterone, and a glucocorticoid.

3. The method of claim 2, wherein the glucocorticoid is hydrocortisone, depomedrol, or dexamethasone.

4. The method of claim 1, wherein the treatment is administered directly to a patient through a mandibular gland, through a thoracic duct, through the lymphatic drainage system.

5. The method of claim 1, wherein the treatment is administered subcutaneously.

6. The method of claim 1, wherein the treatment is administered via a transdermal patch applied to a posterior portion of a patient where lymph vessels of the abdominal wall, pelvic wall, and pelvic limbs drain to lymph nodes.

7. The method of claim 1, wherein the treatment further comprises a non-toxic vehicle, the non-toxic vehicle comprising: a cationic lipid, a neutral lipid, or both; and polyethylene glycol.

8. The method of claim 7, wherein the non-toxic vehicle comprises both the cationic lipid and the neutral lipid at a 3:1 ratio.

9. The method of claim 7, wherein the cationic lipid or the neutral lipid is selected from the group consisting of N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride; dioleoylphosphatidylethanolamine; 2,3-dioleyloxy-N-[2-(spermine-carboxaindo)ethyl]-N,N-dimethyl-1-propanaminium; 5-carboxyspermylglycinedioctadecylaminde; N,N-dimethyl-N-ethylcarboxamidochloesterol; 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine; palmitoyl-sn-glycero-phosphoethanolamine; and 1,2-dimyristoyl-snglycero-3-phospho-ethanolamine.

10. The method of claim 7, wherein the polyethylene glycol is selected from the group consisting of LIPOFECTAMINE, polyethylene glycol 200, polyethylene glycol 300, and polyethylene glycol 1500.

11. The method of claim 1, further comprising: administering hydrocortisone subsequent to, or with, the administration of the treatment.

12. The method of claim 11, further comprising: administering androst-5-ene-3,17-diol or 5-androstene-3,7,17-triol with the hydrocortisone.

13. The method of claim 11, further comprising: administering testosterone, estrogen, or both with the hydrocortisone.

14. An immunomodulator comprising: delta 5-androsten-3B, 17B-diol, delta 5-androsten-3B, 7B, 17B-triol, or both; and a non-toxic vehicle comprising a cationic lipid or a neutral lipid and polyethylene glycol.

15. The immunomodulator of claim 14, further comprising: one or more of estradiol, testosterone, dihydrotestosterone, and a glucocorticoid.

16. The immunomodulator of claim 14, wherein the cationic lipid or the neutral lipid is selected from the group consisting of N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride; dioleoylphosphatidylethanolamine; 2,3-dioleyloxy-N-[2-(spermine-carboxaindo)ethyl]-N,N-dimethyl-1-propanaminium; 5-carboxyspermylglycinedioctadecylaminde; N,N-dimethyl-N-ethylcarboxamidochloesterol; 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine; palmitoyl-sn-glycero-phosphoethanolamine; and 1,2-dimyristoyl-snglycero-3-phospho-ethanolamine.

17. The immunomodulator of claim 14, wherein the polyethylene glycol is selected from the group consisting of LIPOFECTAMINE, polyethylene glycol 200, polyethylene glycol 300, and polyethylene glycol 1500.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the present invention and, together with a general description of the invention given above, and the detailed description of the embodiments given below, serve to explain the principles of the present invention.

[0012] FIGS. 1-3 are a flowcharts illustrating a method of preparing and using an immune system model according to embodiments of the present invention.

[0013] FIG. 4 is an exemplary photograph of 293T cells of normal, distinct phenotype.

[0014] FIG. 5 is an exemplary photograph of 293T cells treated with PEG 300 only.

[0015] FIG. 6 is an exemplary photograph of 293T cells treated with PEG 300 and AED.

[0016] FIG. 7 is an exemplary photograph of 293T cells treated with PEG 300 and 17-estradiol.

[0017] FIG. 8 is an exemplary photograph of 293T cells treated with PEG 300, AED, and 17-estradiol.

[0018] FIG. 9 is an exemplary photograph of 293T cells treated with PEG 300 and LIPOFECTAMINE.

[0019] FIG. 10 is an exemplary photograph of 293T cells treated with PEG 300, LIPOFECTAMINE, and AED.

[0020] FIG. 11 is an exemplary photograph of 293T cells treated with PEG 300, LIPOFECTAMINE, and 17-estradiol.

[0021] FIG. 12 is an exemplary photograph of 293T cells treated with PEG 300, LIPOFECTAMINE, AED, and 17-estradiol.

[0022] FIG. 13 is a graphical representation of a number of mobile cells per field of view of in vitro androstene hormone treatments in 293T cells.

[0023] FIG. 14 is an exemplary photograph of MCF-7 cells treated with a PEG 300 and LIPOFECTAMINE solution, diluted 1:1000.

[0024] FIG. 15 is an exemplary photograph of MCF-7 cells treated with a 3-AED, PEG 300, and LIPOFECTAMINE solution, diluted 1:1000.

[0025] FIG. 16 is an exemplary photograph of MCF-7 cells treated with a -AET, PEG 300, and LIPOFECTAMINE solution, diluted 1:1000.

[0026] FIG. 17 is an exemplary photograph of MCF-7 cells treated with an E2, PEG 300, and LIPOFECTAMINE solution, diluted 1:5000.

[0027] FIG. 18 is an exemplary photograph of MCF-7 cells treated with a -AED, PEG 300, and LIPOFECTAMINE solution, diluted 1:1000, and a solution of E2, PEG 300, and LIPOFECTAMINE, diluted 1:5000.

[0028] FIG. 19 is an exemplary photograph of MCF-7 cells treated with a -AET, PEG 300, and LIPOFECTAMINE solution, diluted 1:1000, and a solution of E2, PEG 300, and LIPOFECTAMINE, diluted 1:5000.

[0029] FIG. 20 is an exemplary photograph of MCF-7 cells treated with a -AET, PEG 300, and LIPOFECTAMINE solution, diluted 1:1000, and a solution of -AET, PEG 300, and LIPOFECTAMINE, diluted 1:1000.

[0030] FIG. 21 is an exemplary photograph of MCF-7 cells treated with a -AED, PEG 300, and LIPOFECTAMINE solution, diluted 1:1000, a solution of -AET, PEG 300, and LIPOFECTAMINE, diluted 1:1000, and a solution of E2, PEG 300, and LIPOFECTAMINE, diluted 1:5000.

[0031] It should be understood that the appended drawings are not necessarily to scale, presenting a somewhat simplified representation of various features illustrative of the basic principles of the invention. The specific design features of the sequence of operations as disclosed herein, including, for example, specific dimensions, orientations, locations, and shapes of various illustrated components, will be determined in part by the particular intended application and use environment. Certain features of the illustrated embodiments have been enlarged or distorted relative to others to facilitate visualization and clear understanding. In particular, thin features may be thickened, for example, for clarity or illustration.

DETAILED DESCRIPTION OF THE INVENTION

[0032] Turning now to the figures, and in particular to FIG. 1, a flow chart 130 illustrating a method of preparing an immune system model according to an embodiment of the present invention is shown. Again, while not necessary, the illustrated embodiments include S. scrofa; however, other appropriate mammals may be used in the alternative as the specimen.

[0033] At start, an immune response is stimulated (Block 132) by injecting a particular antigen into the specimen. Antigens may include commercially-available bacterial, fungal, or viral specific antigens configured to stimulate the formation of protective antibodies (i.e., a thymic dependent response), such as those found in a vaccine.

[0034] Chronic inflammation conditions include a prolonged and extended period of inflammation. The chronic inflammatory response to would related antigens is marked by increased granulocytes, Th1 T cells, M1 macrophages, and a decreased amount of dendritic cells. More particularly, the thymic immune response includes responses to T cell dependent antigenic proteins, which may include animal, bacterial, viral, or fungal proteins. Non-thymic immune responses include non-specific antigens (non-protein, less specific particles) that may illicit a non-specific immune reaction, such as an allergic response or asthma.

[0035] Systemic baseline conditions before and after stimulating the immune response may extend to the brain, skin, thymus, spleen, liver, adrenals, gonads, lymph nodes, small intestine, small intestine mesentery, and circulatory system.

[0036] Continued stimulation generally depends on the system investigated. Generally, 30 days (or roughly four weeks) are necessary for a primary and immune response to generate antibodies from an antigen exposure. A secondary response may be obtained by reinjecting the antigen after the 30 day primary response. Accordingly, the desired response may be achieved by a first stimulation in the first 30 days and, optionally depending on the phenome to be investigated, at least one subsequent stimulation every 30 days (or approximately every four weeks) (Block 134).

[0037] With desired response stimulated, modulation of the immune response may be initiated (Block 136). Immunomodulators include those chemical agents that modify the immune response by stimulating antibody formation or inhibiting white blood cell activity. According to embodiments of the present invention, immunomodulators may include one or more of delta 5-androsten-3B, 17B-diol (.sup.5-diol) and delta 5-androsten-3B, 7B,17B-triol (.sup.5-triol), and further optionally with one or more of estradiol, testosterone, and dihydrotestosterone (DHT).

[0038] If desired, delivery of the immunomodulator may be assisted, according to one embodiment of the present invention, by preparing a non-toxic vehicle (Block 138). Briefly, embodiments of the present invention overcome previous difficulties of steroid hormone solubility and delivery of measureable doses to produce biological effects. Accordingly, and as illustrated in the flow chart 138 of FIG. 2, the non-toxic vehicle includes preparation of a composition comprising a cationic or neutral lipid and polyethylene glycol (PEG) (Block 140). Suitable cationic and neutral lipids may include, for example, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA); dioleoylphosphatidylethanolamine (DOPE); 2,3-dioleyloxy-N-[2-(spermine-carboxaindo)ethyl]-N,N-dimethyl-1-propanaminium (DOSPA); 5-carboxyspermylglycinedioctadecylaminde (DOGS); N,N-dimethyl-N-ethylcarboxamidochloesterol (DC-Chol); 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE); palmitoyl-sn-glycero-phosphoethanolamine (PPE); and 1,2-dimyristoyl-snglycero-3-phospho-ethanolamine (DMPE).

[0039] Cellular membranes generally comprise a phospholipid bilayer such that outer surfaces of the cellular membrane comprise the hydrophilic head groups of the phospholipids with the hydrophobic tails directed centrally between the outer surfaces. Cationic and neutral lipids may assist in trafficking hormone to and across the cellular membrane. Suitable cationic and neutral lipid solutions may include, for example, the commercially-available LIPOFECTAMINE (ThermoFisher Scientific Inc., Waltham, Mass.) or any appropriate PEG formulation in any weight may be used, including but not limited to, PEG 200, PEG 300, PEG 1500, and so forth.

[0040] The liposomal formulation may be prepared in an aqueous solution comprising two solutions. In a first solution, the immunomodulator (-AED (Steraloids, Inc., Newport, R.I.) or -AET (Steraloids, Inc.)) is dissolved in a polyester (for example, PEG), the latter of which may be pre-warmed. In a second solution, a dilution of the cationic and neutral lipid, for example, a 3:1 mixture of cationic to neutral lipid (DOSPA:DOPE) as in LIPOFECTAMINE, may be introduced into a polyester, mixed, and incubated (incubation may be at 42 C. for 30 min). The first and second solutions may then be combined.

[0041] A solution comprising the immunomodulator may then be introduced to the liposomal formulation (Block 142). Evidence of undissolved hormone may include the presence of crystals in in vitro cultures. If crystals are present, then the solution may be warned to above 42 C.

[0042] While not wishing to be bound by theory, it is thought that the cationic liposome formulation provides a solvation and delivery base. While PEG has the ability to, alone, dissolve and deliver hormones, when cationic liposome formulations are used, the PEG solution is diluted, improving solubility, and decreasing toxicity.

[0043] Returning to FIG. 1, the immunomodulator, with or without the treatment vehicle of Block 138, may administered directly or indirectly through the mandibular gland, the thoracic duct, or through the lymphatic drainage system. Alternatively, the immunomodulator may be subcutaneously injected in, or delivered by a transdermal patch applied to, an anterior portion of the patient where lymph vessels of the thoracic limb drain to various lymph nodes (mandibular, parotid, accessory mandibular, lateral retropharyngical, ventral superficial cervical, dorsal superficial cervical, and axillaresprimae costae (costo-axillary)). Alternatively still, the immunomodulator may be subcutaneously injected in, or delivered by a transdermal patch applied to, a posterior portion of the patient where lymph vessels of the abdominal wall, pelvic wall, and pelvic limbs drain to various lymph nodes (subiliac, superficial inguinal, accessory superficial inguinal, superficial popliteal, deep popliteal, external sacral, anterior sacral, medial iliacs, and lateral iliac).

[0044] Generally, only one application (whether subcutaneous injection or transdermal patch) is required for immunomodulation (Yes branch of Decision Block 144). However, it would be appreciated by those having ordinary skill in the art and the benefit of the disclosure made herein that other embodiment of the invention of immunomodulatory schemes or other combinations of immunomodulators may be induced by one subcutaneous injection or transdermal application.

[0045] The initial dosage of immunomodulatory (Block 136), or any subsequent dosage (No branch of Decision Block 144) may require sequential hormone treatments (Block 146) with a repeat dose of the immunomodulatory. For example, hydrocortisone followed by an application of 37 ng/kg or higher of androst-5-ene-3, 17-diol (-AED) or 5-androstene-3,7, 17-triol (-AET). Still other combinations with additional hormones, such as testosterone or estrogen, may be used to achieve a desired method of immunomodulation.

[0046] Referring now to FIG. 3, a flowchart illustrating a method 150 for causing cellular death of triple-negative breast cancer cells by hormone modulation is described according to an embodiment of the present invention is shown. Triple-negative breast cancer comprises a heterogeneous subset of breast cancer-types. While accounting for 15% to 25% of all breast cancer cases, the triple negative cancers are not supported by estrogen and progesterone because the cells are ER and PR receptor negative and have an increased number of HER2 receptors.

[0047] At start, a combination of adrenal hormones, including 5-androsten-3,17-diol and 5-androsten-3,7,17-triol (both at a concentration of about 25 M) with or without estradiol (E2) is prepared (at a concentration of about 5 nM) (Block 152). The adrenal hormone solutions may be prepared and administered separately or together.

[0048] Optionally, the preparation may include a suitable vehicle (Block 154), such as PEG, ethanol, dimethylsulfoxide (DMSO), or the PEG-LIPOFECTAMINE vehicle according to other embodiments of the present invention, for example as described with reference to FIG. 2. In such embodiments, the respective adrenal hormone solution may be mixed with the cationic or neutral lipid and polyethyleneglycol.

[0049] In vitro treatment may include applying the adrenal hormones with vehicle to a cell growth medium. In vivo treatment may be injected directly into a tumor, applied to an area from which a tumor was surgically removed, or injected subcutaneously.

[0050] Only one application (whether subcutaneous injection or transdermal patch) is required for immunomodulation (Yes branch of Decision Block 156). However, it would be appreciated by those having ordinary skill in the art and the benefit of the disclosure made herein that other embodiment of the invention of immunomodulatory schemes or other combinations of immunomodulators may be induced by one subcutaneous injection or transdermal application.

[0051] The initial dosage of immunomodulatory (Block 154), or any subsequent dosage (No branch of Decision Block 156) may require sequential hormone treatments (Block 158) with a repeat dose of the immunomodulatory. For example, hydrocortisone, testosterone, or estrogen, may be used to achieve a desired method of immunomodulation.

[0052] Although not specifically illustrated herein, it would be understood that the various embodiments of the treatment vehicle described herein need not be limited to delivery of an immunomodulator or in the modeling of an immune response. Accordingly, the treatment vehicle may be used, when appropriate or desired, to achieve delivery of hormone, steroid, or the like to achieve a desired physiological effect that would otherwise difficult to achieve.

[0053] Preparation and delivery of hormone in according to embodiments as describe herein provide the benefit in that the treated cultures are not morphologically or phenotypically changed as compared to control cells. Full dissolution of the hormone reduces toxicity and reduces interference with physiological effects of the hormones. Moreover, use of the non-toxic vehicle reduces component residue at the point of application, allowing for more precise measurement of dosage administration.

[0054] The following examples illustrate particular properties and advantages of some of the embodiments of the present invention. Furthermore, these are examples of reduction to practice of the present invention and confirmation that the principles described in the present invention are therefore valid but should not be construed as in any way limiting the scope of the invention.

Example 1

[0055] A non-toxic vehicle for administration of androstene hormones according to embodiments of the present invention was prepared from a first solution and a second solution. The first solution, comprising liposomal formulation, was placed in PEG 300 to a concentration of 5% and heated in a sonicating water bath to about 45 C. The second solution, comprising a selected hormone (one or more of 5-androsten-3, 17-diol, 5-androsten-3, 17-diol, or 5-androsten-3,7,17-triol), was placed in PEG 300 and heated in a sonicating water bath to about 45 C. The first and second solutions were then combine and stirred until dissolved.

[0056] Biological function was tested in 293T cells, which is a human embryonic cell line transformed with Large T antigen or SV40. This hypotriploid (polyploid) cell line is particularly useful in such transformation studies and has very distinct phenotypes when grown. All androstene hormone treatment applications were at concentrations of 25 M.

[0057] FIG. 4 is an exemplary photograph of 293T cells of normal, distinct phenotype. There are small cell islands, large cell islands, and mobile cells/cell processes.

[0058] FIG. 5 is an exemplary photograph of 293T cells treated with PEG 300 only. The cells demonstrate the same characteristics as the normal cells of FIG. 3.

[0059] FIG. 6 is an exemplary photograph of 293T cells treated with PEG 300 and AED. Resultant cell islands are larger and there is a significant reduction in mobile cells.

[0060] FIG. 7 is an exemplary photograph of 293T cells treated with PEG 300 and 17-estradiol. The culture includes many more cell processes as compared to estradiol alone.

[0061] FIG. 8 is an exemplary photograph of 293T cells treated with PEG 300, AED, and 17-estradiol. There is a visible decrease in mobile cells over the treatment with AED alone.

[0062] FIG. 9 is an exemplary photograph of 293T cells treated with PEG 300 and LIPOFECTAMINE, the latter of which increased granularity. Otherwise, cells were similar to the Blanks, despite presence of PEG 300.

[0063] FIG. 10 is an exemplary photograph of 293T cells treated with PEG 300, LIPOFECTAMINE, and AED. The treatment combination demonstrated a drastic decrease in, or morphological change, in mobile elements.

[0064] FIG. 11 is an exemplary photograph of 293T cells treated with PEG 300, LIPOFECTAMINE, and 17-estradiol. The combination produced many large islands with mobile processes, small islands with mobile process, and mobile cells forming interconnections with other elements.

[0065] FIG. 12 is an exemplary photograph of 293T cells treated with PEG 300, LIPOFECTAMINE, AED, and 17-estradiol. As compared to cells of FIG. 11, mobile cells and processes are largely non-existent.

[0066] FIG. 13 is a graphical representation of a number of mobile cells per field of view of in vitro androstene hormone treatments in 293T cells, described above, wherein column A is a blank, column B is PEG 300 with LIPOFECTAMINE, column C is PEG 300 with LIPOFECTAMINE with 25 M -AED, column D is PEG 300 with LIPOFECTAMINE with 5 nM E2, column E is PEG 300 with LIPOFECTAMINE with 25 M -AED and 5 nM E2, column F is PEG 300 with LIPOFECTAMINE, column G is PEG 300 with LIPOFECTAMINE and 5 nM E2, column H is PEG 300 with LIPOFECTAMINE and 25 M -AED, and column I is PEG 300 with LIPOFECTAMINE, 25 M -AED, and 5 nM E2.

Example 2

[0067] Hormone dependent, noninvasive, epithelial phenotype (ER/PR positive MCF-7 breast cancer cells) and hormone independent, invasive, mesenchymal phenotype (ER/PR negative MDA-MB-231 breast cancer cell) cell suspensions were acquired and prepared in complete medium (DMEM/F12, 10% FBS, 1% penicillin/streptomycin (Gibco)). Cellular concentration was adjusted to 210.sup.5 cells/mL.

[0068] Androstene hormone solutions according to embodiments of the present invention were prepared from a first solution and a second solution. For the first solution, 3.0 mg of -AED or 3-AET was added to warm PEG 300 to prepare 50 mM stock. For the second solution, 10 L of LIPOFECTAMINE (3:1 DOSPA:DOPE) was introduced to 100 L of PEG 300, mixed, and incubated in a water bath at 42 C. for 30 min. The first and second solutions were then mixed in a sonicator water bath at 42 C. for 1 hr. The final concentration was 50 mM -AED (or AET, as used).

[0069] A -estradiol solution was also prepared by adding 2.7 mg of -estradiol to 200 L warmed PEG 300 with LIPOFECTAMINE and then further diluted 1:1000 with PEG-300. The final concentration was 50 M.

[0070] A diluted -AED (or 3-AET) solution was prepared with complete medium at a final concentration of 50 M (1:1000 dilute of 50 mM). Likewise, a diluted -E2 solution was prepared with complete medium at a final concentration of 10 nM (1:5000 dilute of 50 M).

[0071] Using a 24-well plate, mixtures of 0.5 mL of cells per well with 0.5 mL of medium diluted hormone vehicle. Cell cultures were maintained at 90% humidity, 7% CO.sub.2, and 37 C.

[0072] Results are provided in tabular format below:

TABLE-US-00001 TABLE 1 MDA-MB-231 MCF7 Treatment Cell Death Amount Cell Death Amount Additional Observation -AED Not observed Not observed Cell particles observed -AED + E2 70% Not observed Cell particles observed -AED + -AET 80% Not observed Cell particles observed -AED + -AET + E2 90% Not observed Cell particles observed -AET Not observed Not observed N/A E2 Not observed Not observed N/A

[0073] The MDA-MB-231 cell cultures treated with (1) 25 M -AED and 5 nM E2, (2) 25 M -AED and 25 M AET, or (3) 25 M -AED, 25 M AET and 5 nM E2 yielded cell death rates of 70%, 80%, and 90%, respectively. Cell death was not observed in MCF7 cell cultures treated with the same doses.

[0074] FIG. 14 is a microscopic photograph of MCF-7 cells grown for 5 days in a cell medium comprising PEG 300 with 5% LIPOFECTAMINE solution diluted 1:1000.

[0075] FIG. 15 is a microscopic photograph of MCF-7 cells grown for 5 days in a cell medium comprising 25 mM stock solution of -AED in PEG 300 with 5% LIPOFECTAMINE solution diluted 1:1000 to a concentration of 25 M. As compared to FIG. 14, the cell density of FIG. 15 is not as heath and there are areas of decreased density. FIG. 15 also presents small particles (identified by arrows) that were produced by cells in response to the treatment with -AED.

[0076] FIG. 16 is a microscopic photograph of MCF-7 cells grown for 5 days in a cell medium comprising 25 mM stock solution of 3-AET in PEG 300 with 5% LIPOFECTAMINE solution diluted 1:1000 to a concentration of 25 M. The cell growth is thick and similar to the cell growth of FIG. 14.

[0077] FIG. 17 is a microscopic photograph of MCF-7 cells grown for 5 days in a cell medium comprising 25 M solution of E2 in PEG 300 with 5% LIPOFECTAMINE solution diluted 1:5000 to a concentration of 5 nM. The cell growth is thick and similar to the cell growth of FIGS. 14 and 16.

[0078] FIG. 18 is a microscopic photograph of MCF-7 cells grown for 5 days in a cell medium comprising 25 mM stock solution of -AED in PEG 300 with 5% LIPOFECTAMINE solution diluted 1:1000 to a concentration of 25 M and 25 M stock solution of E2 in PEG 300 with 5% LIPOFECTAMINE solution diluted 1:5000 to a concentration of 5 nM. The cell growth is thick and similar to the cell growth of FIGS. 14, 16, and 17.

[0079] FIG. 19 is a microscopic photograph of MCF-7 cells grown for 5 days in a cell medium comprising 25 mM stock solution of 3-AET in PEG 300 with 5% LIPOFECTAMINE solution diluted 1:1000 to a concentration of 25 M and 25 M stock solution of E2 in PEG 300 with 5% LIPOFECTAMINE solution diluted 1:5000 to a concentration of 5 nM. The cell growth is thick and similar to the cell growth of FIGS. 14 and 16-18.

[0080] FIG. 20 is a microscopic photograph of MCF-7 cells grown for 5 days in a cell medium comprising 25 mM stock solution of -AED in PEG 300 with 5% LIPOFECTAMINE solution diluted 1:1000 to a concentration of 25 M and 25 mM stock solution of -AED in PEG 300 with 5% LIPOFECTAMINE solution diluted 1:1000 to a concentration of 25 M. The cell growth is thick and similar to the cell growth of FIGS. 14 and 16-19.

[0081] FIG. 21 is a microscopic photograph of MCF-7 cells grown for 5 days in a cell medium comprising 25 mM stock solution of -AED in PEG 300 with 5% LIPOFECTAMINE solution diluted 1:1000 to a concentration of 25 M, 25 mM stock solution of -AED in PEG 300 with 5% LIPOFECTAMINE solution diluted 1:1000 to a concentration of 25 M, and 25 M stock solution of E2 in PEG 300 with 5% LIPOFECTAMINE solution diluted 1:5000 to a concentration of 5 nM. While the figure presents a full growth of cells, the cell layer is not as thick as was presented in FIGS. 14 and 16-19. The treatment caused the cells to produce small cell bodies.

[0082] While the present invention has been illustrated by a description of one or more embodiments thereof and while these embodiments have been described in considerable detail, they are not intended to restrict or in any way limit the scope of the appended claims to such detail. Additional advantages and modifications will readily appear to those skilled in the art. The invention in its broader aspects is therefore not limited to the specific details, representative apparatus and method, and illustrative examples shown and described. Accordingly, departures may be made from such details without departing from the scope of the general inventive concept.