METHOD OF TREATING MELANOMA USING AN INHIBITOR OF AN ATYPICAL PROTEIN KINASE C
20170348336 · 2017-12-07
Assignee
Inventors
Cpc classification
C12Q2600/106
CHEMISTRY; METALLURGY
A61K31/194
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
A61K31/185
HUMAN NECESSITIES
A61K31/675
HUMAN NECESSITIES
A61K31/122
HUMAN NECESSITIES
G01N2800/52
PHYSICS
International classification
A61K31/675
HUMAN NECESSITIES
A61K31/122
HUMAN NECESSITIES
A61K31/185
HUMAN NECESSITIES
Abstract
The invention pertains to a method of treating melanoma by administering to a subject in need thereof, a composition comprising a therapeutically effective amount of an inhibitor of PKC-ζ and/or PKC-.Math.. Non-limiting examples of an inhibitor of PKC-ζ and/or PKC-.Math. include ICA-1 and ACPD. The invention also provides PKC-ζ and/or PKC-.Math. as biomarkers for identifying a melanoma in a subject as likely to be responsive or non-responsive to a therapy using an inhibitor of PKC-ζ and/or PKC-.Math.. Accordingly, a method of identifying a subject having a melanoma as being responsive or non-responsive to a melanoma therapy with an inhibitor of PKC-ζ and/or PKC-.Math. based on the levels and/or activity of PKC-ζ and/or and/or PKC-.Math. mRNA or protein in the melanoma cells from the subject are also provided.
Claims
1. A method of treating a melanoma in a subject in need thereof, the method comprising administering a therapeutically effective amount of an inhibitor of an atypical protein kinase C (aPKC).
2. The method of claim 1, wherein the aPKC is PKC-iota (PKC-.Math.) or PKC-zeta (PKC-ζ).
3. The method of claim 1, wherein the PKC inhibitor is specific to a single aPKC.
4. The method of claim 3, wherein the aPKC is PKC-iota.
5. The method of claim 4, wherein the aPKC inhibitor is 1H-imidazole-4-carboxamide, 5-amino-1-[2,3-dihydroxy-4-[(phosphonooxy)methyl]cyclopentyl]-[1R-(1.alph-a., 2.beta., 3.beta., 4.alpha.)] (ICA-1).
6. The method of claim 1, wherein the aPKC inhibitor is a pan-aPKC inhibitor.
7. The method of claim 6, wherein the aPKC inhibitor is 2-acetyl-1,3-cyclopentanedione (ACPD).
8. The method of claim 3, wherein the aPKC is PKC-zeta.
9. The method of claim 8, wherein the aPKC inhibitor is 8-hydroxy-1, 3,6-naphthalenetrisulfonic acid (ζ-Stat).
10. A method of inhibiting a melanocyte proliferation, the method comprising contacting the melanocyte with a therapeutically effective amount of an inhibitor of an aPKC.
11. The method of claim 10, wherein the aPKC is PKC-iota or PKC-zeta.
12. The method of claim 10, wherein the PKC inhibitor is specific to a single aPKC.
13. The method of claim 12, wherein the aPKC is PKC-iota.
14. The method of claim 13, wherein the PKC inhibitor is ICA-1.
15. The method of claim 10, wherein the aPKC inhibitor is a pan-aPKC inhibitor.
16. The method of claim 15, wherein the aPKC inhibitor is ACPD.
17. The method of claim 12, wherein the aPKC is PKC-zeta.
18. The method of claim 17, wherein the PKC inhibitor is zeta-Stat (ζ-Stat).
19. A method of reducing levels of at least one aPKC in melanocytes, the method comprising contacting the melanocytes with a therapeutically effective amount of an inhibitor of an aPKC.
20. The method of claim 19, wherein the aPKC is PKC-iota or PKC-zeta.
21. The method of claim 19, wherein the aPKC inhibitor is a pan-aPKC inhibitor.
22. The method of claim 19, wherein the aPKC inhibitor is specific to a single aPKC.
23. The method of claim 20, wherein the aPKC inhibitor is ICA-1 and ACPD.
24. The method of claim 20, wherein the aPKC inhibitor is zeta-Stat (ζ-Stat).
25. A method of identifying a subject suffering from a melanoma as responsive or non-responsive to a therapy with an inhibitor of PKC-ζ and/or PKC-.Math., the method comprising the steps of: (a) determining the level and/or activity of PKC-ζ and/or PKC-.Math. mRNA or protein in a test sample obtained from the subject, (b) optionally, determining the level and/or activity of PKC-ζ and/or PKC-.Math. mRNA or protein in a control sample, or obtaining a reference value corresponding to the level of PKC-ζ, and/or PKC-.Math. mRNA or protein; and (i) identifying the subject as being responsive to the melanoma therapy with the inhibitor of PKC-ζ and/or PKC-.Math. based the level and/or activity of PKC-ζ and/or PKC-.Math. mRNA or protein in the test sample as compared to the level and/or activity of PKC-ζ and/or PKC-.Math. mRNA or protein in the control sample, and optionally administering the melanoma therapy with the inhibitor of PKC-ζ and/or PKC-.Math. to the subject identified as being responsive; or (ii) identifying the subject as being non-responsive to the melanoma therapy with the inhibitor of PKC-ζ and/or PKC-.Math. based the level and/or activity of PKC-ζ and/or PKC-.Math. mRNA or protein in the test sample as compared to the level and/or activity of PKC-ζ and/or PKC-.Math. mRNA or protein in the control sample, and optionally administering a melanoma therapy other than the melanoma therapy with the inhibitor of PKC-ζ and/or PKC-.Math. to the subject identified as being non-responsive.
26. The method of claim 25, wherein, the test sample comprises the melanoma cells from the subject.
27. The method of claim 25, wherein: i) the subject having the melanoma is identified as being responsive to the melanoma therapy with the inhibitor of PKC-ζ and/or PKC-.Math. if the melanoma cells in the subject contain a higher level and; or activity of mRNA or protein for PKC-ζ and; or PKC-.Math. in the melanoma cells from the subject compared to a control sample of non-melanoma cells; and ii) the subject having the melanoma is identified as being non-responsive to the melanoma therapy with the inhibitor of PKC-ζ and/or PKC-.Math. if the melanoma cells in the subject contain a similar or lower level and/or activity of mRNA or protein for PKC-ζ and/or PKC-.Math. in the melanoma cells from the subject compared to the control sample of non-melanoma cells.
28. The method of claim 25, wherein the control sample is one or more of the following: a) melanocytes from an individual belonging to the same species as the subject and not having melanoma, b) melanocytes from the subject prior to getting melanoma, c) melanocytes from the subject not affected by melanoma, d) melanoma cells from an individual known to be responsive to a melanoma therapy with an inhibitor of PKC-ζ and/or PKC-.Math., or e) melanoma cells from an individual known to be non-responsive to a melanoma therapy with an inhibitor of PKC-ζ and/or PKC-.Math..
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DISCLOSURE OF THE INVENTION
[0058] As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Further, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising”. The transitional terms/phrases (and any grammatical variations thereof) “comprising”, “comprises”, “comprise”, “consisting essentially of”, “consists essentially of”, “consisting” and “consists” can be used interchangeably.
[0059] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 0-20%, 0 to 10%, 0 to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed. In the context of compositions containing amounts of ingredients where the terms “about” or “approximately” are used, these compositions contain the stated amount of the ingredient with a variation (error range) of 0-10% around the value (X±10%).
[0060] “Pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the PKC inhibitors described herein, its use in the compositions of the invention is contemplated.
[0061] “Treatment”, “treating”, “palliating” and “ameliorating” (and grammatical variants of these terms), as used herein, are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results including but not limited to therapeutic benefit. A therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying cancer such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying cancer. A treatment includes delaying the appearance of a disease or condition, delaying the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.
[0062] As used herein, the term “cancer” refers to the presence of cells possessing abnormal growth characteristics, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, perturbed oncogenic signaling, and certain characteristic morphological features. This includes but is not limited to the growth of: (1) benign or malignant cells (e.g., tumor cells) that correlates with overexpression of a serine/threonine kinase; or (2) benign or malignant cells (e.g., tumor cells) that correlates with abnormally high levels of serine/threonine kinase activity or lipid kinase activity. Non-limiting serine/threonine kinases implicated in cancer include but are not limited to PI-3K mTOR, and AKT. Exemplary lipid kinases include but are not limited to PI3 kinases such as PBKα, PBKβ, PBKδ, and PBKγ.
[0063] The term “effective amount” or “therapeutically effective amount” refers to that amount of an inhibitor described herein that is sufficient to effect the intended application including but not limited to disease treatment. The therapeutically effective amount may vary depending upon the intended application (in vitro or in vivo), or the subject and disease condition being treated, e.g., the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The term also applies to a dose that will induce a particular response in target cells, e.g., reduction of proliferation or downregulation of activity of a target protein. The specific dose will vary depending on the particular compounds chosen, the dosing regimen to be followed, whether it is administered in combination with other compounds, timing of administration, the tissue to which it is administered, and the physical delivery system in which it is carried.
[0064] For the specific PKC-.Math. inhibitor ICA-1, the therapeutically effective concentration can be between about 800 nM to about 10 μM. In some embodiments the range of ICA-1 concentration is between about 1 μM to about 2 μM. In some embodiments, the therapeutically effective concentration of ICA-1 is about 2 μM.
[0065] For ACPD, the therapeutically effective concentration can be between about 800 nM to about 10 μM. In some embodiments, the therapeutically effective concentration was found to be about 2 μM. Therapeutically effective concentrations for other PKC-.Math. inhibitors can be readily determined by those of ordinary skill in the art.
[0066] The amount of ICA-1 or ACPD administered can be an amount from a low of about 0.8 μM, about 0.9 μM, or about 1 μM to a high of about 2 μM, about 5 μM, or about 10 μM. For example, the amount of ICA-1 or ACPD of the subject invention that inhibits growth of melanoma cells can be from about 0.8 μM to about 10 μM, from about 0.9 μM to about 5 μM, from about 1 μM to about 2 μM, from about 0.8 μM to about 5 μM, from about 0.8 μM to about 2 μM, from about 0.9 μM to about 10 μM, from about 0.9 μM to about 5 μM, from about 0.9 μM to about μM, from about 1 μM to about 10 μM, from about 1 μM to about 5 μM, from about 1 μM to about 2 μM, from about 1.5 μM to about 10 μM, from about 1.5 μM to about 5 μM, from about 1.5 μM to about 2 μM, from about 1.8 μM to about 10 μM, from about 1.8 μM to about 5 μM, from about 1.8 μM to about 2 μM.
[0067] For ζ-Stat, the therapeutically effective concentration can be between about 800 nM to about 20 μM. In some embodiments the range of ζ-Stat concentration is between about 1 μM to about 10 μM. In some embodiments, the therapeutically effective concentration of ζ-Stat is about 5 μM.
[0068] The amount of -Stat administered can be an amount from a low of about 0.8 μM, about 0.9 μM, or about 1 μM to a high of about 2 μM, about 5 μM, about 10 μM, or about 20 μM. For example, the amount of -Stat of the subject invention that inhibits growth of melanoma cells can be from about 0.8 μM to about 20 μM, from about 0.9 μM to about 10 μM, from about 1 μM to about 5 μM, from about 0.8 μM to about 10 μM, from about 0.8 μM to about 5 μM, from about 0.8 μM to about 2 μM, from about 0.9 μM to about 20 μM, from about 0.9 μM to about 5 μM, from about 0.9 μM to about 2 μM, from about 1 μM to about 20 μM, from about 1 μM to about 10 μM, from about 1 μM to about 2 μM, from about 1.5 μM to about 20 μM, from about 1.5 μM to about 10 μM, from about 1.5 μM to about 5 μM, from about 1.5 μM to about 2 μM, from about 1.8 μM to about 20 μM, from about 1.8μM to about 10 μM, from about 1.8 μM to about 5 μM, from about 1.8 μM to about 2 μM.
[0069] A “sub-therapeutic amount” of a PKC inhibitor is an amount less than the effective amount for that inhibitor, but which when combined with an effective or sub-therapeutic amount of another therapeutic compound or therapy can produce a desired result, due to, for example, synergy in the resulting efficacious effects (e.g., therapeutic benefit) for the subject, or reduced side effects associated with the compounds administered to the subject. Typical therapeutic amounts for an agent, as disclosed herein, can be ascertained from various publicly available sources (e.g., drugs.com, The Physician's Desk Reference, or scientific literature). Subtherapeutic amounts of an inhibitor, as provided herein, are amounts less than those reported in the publicly available sources.
[0070] “Subject” refers to an animal, such as a mammal, for example a human. The methods described herein can be useful in both humans and non-human animals. In some embodiments, the subject is a mammal (such as an animal model of disease), and in some embodiments, the subject is human. The terms “subject” and “subject” can be used interchangeably.
[0071] The terms “simultaneous” or “simultaneously” as applied to administering therapies to a subject refer to administering one or more therapies at the same time, or at two different time points that are separated by no more than 30 minutes. The term “after or before” as applied to administering therapies to a subject refers to administering more than one doses at two different time points that are separated by more than 30 minutes, e.g., about 1 hour, about 2 hours, about 5 hours, 8 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days or even longer.
[0072] The term “inhibitor” refers to a compound having the ability to inhibit a biological function of a target protein, whether by inhibiting the activity or expression of the target protein. Accordingly, the term “inhibitor” is defined in the context of the biological role of the target protein.
[0073] The term “PKC inhibitor” as used herein refers to an agent that inhibits the activity or reduces/inhibits the expression of one or more isoforms of PKC. Examples of such inhibitors include, but are not limited to, ICA-1, ACPD, ζ-Stat, fludarabine and derivatives thereof; aurothioglucose; aurothiomaleate; auranofin; thimerosal; phenylmercuric acetate; ebselen; cisplatin; taxol; apomorphine; pyrantel pamoate; gossypolacetic acid complex; ellagic acid; hexestrol; Pachastrissamine and its stereoisomers; ZIP; allosteric inhibitors which bind the PIF-1 site and regulate activity through the C1 domain; a series of phenylthiopenes; a compound containing a maleimide substructure; speciosterosulfates (sterolsulfates); a series of compounds having an indazole-benzimidazole motif; a series of ATP-competitive thieno[2,3-d]pyrimidine analogues; a pyrrole amide PKCzI257.3; and a series of 3-hydroxy-2-(3-hydroxyphenyl)-4H-1-benzopyran-4-ones which may bind to the ATP-cleft of the kinase through a keto-hydroxyl motif in multiple conformations; and derivatives thereof. Numbers in parentheses refer to the corresponding numbered structure of the agent listed in the Background section of the application. Certain examples of PKC inhibitors that can be used in the methods described herein are provided in the U.S. Pat. No. 9,301,965.
[0074] The term “pan-aPKC inhibitor” as used herein refers to an agent that inhibits the activity or reduces/inhibits the expression of at least one atypical PKC such as PKC-.Math. or PKC-ζ. Examples of such agents include, but are not limited to ACPD, pachastrissamine and its stereoisomers, and derivatives thereof.
[0075] The terms “PKC-.Math. inhibitor” as used herein refers to an agent that inhibits PKC-.Math. activity or reduces or inhibits expression of PKC-.Math.. The agent may be specific to PKC-.Math. or alternatively may be a pan-aPKC inhibitor that is effective against different aPKC isoforms, such as ACPD. The inhibitor can be a polypeptide that binds to a unique sequence in the catalytic domain of PKC-.Math. and inhibits its activity; a polypeptide that is involved with the interaction of PKC-.Math. with other signaling molecules; a polypeptide having sequence homology to a specific region of a signaling molecule that mediates the binding of these molecules to PKC-.Math.; or a small molecule inhibitor, such as ICA-1 and derivatives thereof.
[0076] The term “PKC-ζ inhibitor” as used herein refers to an agent that inhibits PKC-ζ activity or reduces or inhibits expression of PKC-ζ. The agent may be specific to PKC-ζ, such as ζ-Stat, or alternatively may be a pan-aPKC inhibitor that is effective against different aPKC isoforms, such as ACPD. The inhibitor can be a polypeptide that binds to a unique sequence in the catalytic domain of PKC-ζ and inhibits its activity; a polypeptide that is involved with the interaction of PKC-ζ with other signaling molecules; a polypeptide having sequence homology to a specific region of a signaling molecule that mediates the binding of these molecules to PKC-ζ; or a small molecule inhibitor. Examples of a PKC-ζ inhibitor include, but are not limited to, ZIP, ACPD, speciosterosulfates (sterolsulfates), and derivatives thereof.
[0077] In specific preferred embodiments of the subject invention, the novel atypical PKC inhibitor is specific to PKC-ζ. In more preferred embodiments, the novel atypical PKC inhibitor is 8-hydroxy-1, 3,6-naphthalenetrisulfonic acid (c-Stat).
[0078] “Administration” or “administering” is used to describe the process in which the PKC inhibitors of the present invention are delivered to a subject for treatment. This includes parental, referring to parenterally (intramuscularly, intraperitoneally, intraarterially, intravenously, subcutaneously), orally, topically, transdermally, or vaginally and other routes that allow the PKC inhibitor to contact the cancer cells. The PKC inhibitor may be administered independently or in combination with other compounds, such as other chemotherapeutic compounds.
[0079] The term “pharmaceutically acceptable salt” refers to salts derived from a variety of organic and inorganic counter ions well known in the art. Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, specifically such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. In some embodiments, the pharmaceutically acceptable base addition salt is chosen from ammonium, potassium, sodium, calcium, and magnesium salts.
[0080] As noted above, the number of melanoma cases has increased every year. It is not totally understood which intracellular chemicals are involved in signaling pathways and that govern the metastasis of melanoma cancer cells. Even though PKC-.Math. was not reported in normal melanocytes, it was detected in exclusively high amounts in both transformed melanocytes and melanoma metastases. PKC-.Math. was also reported in both normal melanocytes and melanoma metastases.
[0081] The methods disclosed in the subject invention disclose method to assess the effectiveness of ACPD on both PKC-.Math. and PKC-ζ ICA-1 as an inhibitor of PKC-.Math., and ζ-Stat as inhibitor of PKC-ζ.
[0082] In some embodiments, SK-MEL-2 metastasis melanoma cells, MeWo metastasis melanoma cells and PCS-200-013 normal melanocyte cells are cultivated and treated with ACPD, ICA-1, and/or ζStat in separate sets of flasks for three consecutive days while taking the cell count for every 24 hrs. In certain embodiment, statistically significant decreases in cell number in SK-MEL-2 cells and MeWo cells are observed while no change is observed in PCS-200-013.
[0083] In other embodiments of the subject invention, a scratch assay to test the degree of metastasis and western blotting analysis using cell lysates are provided to test the levels of PKC-.Math. and PKC-ζ. In said assays, the cell population of melanoma cells has an inversely proportional relationship with the drug concentrations. Therefore, an inhibitor of PKC-.Math. and/or PKC-ζ for example, ACPD, ICA-1, or ζ-Stat, decreases the cell population and motility of melanoma cancer cells and thus, can be used in the treatment of melanoma.
[0084] Accordingly, an embodiment of the invention provides a method of treating melanoma, the method comprising administering to a subject in need thereof, a therapeutically effective amount of an inhibitor of an aPKC, for example, PKC-.Math. and PKC-ζ. In one embodiment, the PKC inhibitor is specific to a single aPKC, for example, PKC-.Math..
[0085] In certain embodiments, the specific inhibitor of PKC-.Math. is ICA-1. In other embodiments, the PKC inhibitor is a pan-aPKC inhibitor. In preferred embodiments, the pan-PKC inhibitor is ACPD.
[0086] In certain embodiments, the specific inhibitor of PKC-ζ is ζ-Stat.
[0087] In further embodiments, methods of inhibiting melanocyte proliferation are provided. In preferred embodiments, the methods comprise contacting the melanocyte with a therapeutically effective amount of an inhibitor of an aPKC, for example, PKC-.Math. and PKC-ζ.
[0088] In some embodiments, the PKC inhibitor is specific to a single aPKC, for example, PKC-.Math.. In a preferred embodiment, the specific inhibitor of PKC-.Math. is ICA-1.
[0089] In other embodiments, the PKC inhibitor is a pan-aPKC inhibitor. In preferred embodiments, the pan-aPKC inhibitor is ACPD.
[0090] In yet other embodiments, the PKC inhibitor is specific to a single aPKC, for example, PKC-ζ. In a preferred embodiment, the specific inhibitor of PKC-ζ is ζ-Stat.
[0091] Advantageously, it was determined that ζ-Stat interacts with amino acid residues of 251-547 (I251, R253, V259, K274, D544, and F547) of the catalytic domain of PKC-ζ and that ζ-Stat is specific only to PKC-ζ.
[0092] In further embodiments, aPKC, including, but not limited to, PKC-.Math. and/or PKC-ζ, can be used as a biomarker for identifying a melanoma subject as being responsive to a treatment with an aPKC inhibitor.
[0093] In some embodiment, melanoma cells from a subject having melanoma exhibit a higher expression and/or amount of mRNA or protein for PKC-ζ and/or PKC-.Math.. In preferred embodiments, the melanoma cells of said subject are responsive to a melanoma therapy with an inhibitor of aPKC.
[0094] In a specific embodiment, the subject invention provides a method for identifying a subject as responsive or non-responsive to a melanoma therapy with an inhibitor of an aPKC based on higher expression and/or amount of mRNA or protein for PKC-ζ and/or PKC-.Math. in melanoma cells from the subject compared to a control.
[0095] The phrase “a subject is responsive to a melanoma therapy” indicates that upon administration of the melanoma therapy, the subject exhibits beneficial or desired results including but not limited to therapeutic benefit; eradication, amelioration, delay or prevention of one or more of the symptoms of melanoma; prolonging the life-span of the subject; or improvement in the quality of life of the subject. An improvement may be observed in the patient, notwithstanding that the patient may still be afflicted with melanoma.
[0096] The phrase “a subject is non-responsive to a melanoma therapy” indicates that upon administration of the melanoma therapy, the subject does not exhibit beneficial or desired results such as, therapeutic benefit; eradication, amelioration, delay or prevention of one or more of the symptoms of melanoma; prolonging the life-span of the subject; or improvement in the quality of life of the subject.
[0097] It was advantageously determined that a subject having a melanoma wherein the melanoma cells from the subject exhibit a higher level and/or activity of mRNA or protein for PKC-ζ and/or PKC-.Math. is responsive to a melanoma therapy with an inhibitor of PKC-ζ and/or PKC-.Math..
[0098] Accordingly, in preferred embodiments of the subject invention, methods are provided for identifying a subject as responsive or non-responsive to a melanoma therapy with an inhibitor of PKC-ζ and/or PKC-.Math. based on higher level and/or activity of mRNA or protein for PKC-ζ and/or PKC-.Math. in the melanoma cells from the subject compared to a control.
[0099] In a certain embodiment, the subject invention provides a method of identifying a subject suffering from a melanoma as responsive or non-responsive to a therapy with an inhibitor of PKC-ζ and/or PKC-.Math.. The method comprises the steps of:
[0100] (a) determining the level and/or activity of PKC-ζ and/or PKC-.Math. mRNA or protein in a test sample obtained from the subject,
[0101] (b) optionally, determining the level and/or activity of PKC-ζ and/or PKC-.Math. mRNA or protein in a control sample, or obtaining a reference value corresponding to the level of PKC-ζ and/or PKC-.Math. mRNA or protein; and
[0102] (i) identifying the subject as being responsive to the melanoma therapy with the inhibitor of PKC-ζ and/or PKC-.Math. based the level and/or activity of PKC-ζ and/or PKC-.Math. mRNA or protein in the test sample as compared to the level and/or activity of PKC-ζ and/or PKC-.Math. mRNA or protein in the control sample, and optionally, administering the melanoma therapy with the inhibitor of PKC-ζ and/or PKC-.Math. to the subject identified as being responsive; or
[0103] (ii) identifying the subject as being non-responsive to the melanoma therapy with the inhibitor of PKC-ζ and/or PKC-.Math. based the level and/or activity of PKC-ζ and/or PKC-.Math. mRNA or protein in the test sample as compared to the level and/or activity of PKC-ζ and/or PKC-.Math. mRNA or protein in the control sample, and optionally, administering a melanoma therapy other than the melanoma therapy with the inhibitor of PKC-ζ and/or PKC-.Math. to the subject identified as being non-responsive.
[0104] In specific embodiments, the test samples comprise the melanoma cells from the subject.
[0105] It was advantageously determined that a subject's melanoma is responsive to a melanoma therapy with an inhibitor of PKC-ζ and/or PKC-.Math. if the melanoma cells in the subject contain a higher level and/or activity of mRNA or protein for PKC-ζ and/or PKC-.Math. in the melanoma cells from the subject compared to a control sample of non-melanoma cells.
[0106] It was also determined that a subject's melanoma is non-responsive to a melanoma therapy with an inhibitor of PKC-ζ and/or PKC-.Math. if the melanoma cells in the subject contain a similar or lower level and/or activity of mRNA or protein for PKC-ζ and/or PKC-.Math. in the melanoma cells from the subject compared to a control sample of non-melanoma cells.
[0107] Various techniques are known to a person of ordinary skill in the art to determine the level of mRNA in a sample. Non-limiting examples of such techniques include microarray analysis, real-time polymerase chain reaction (PCR), Northern blot, in situ hybridization, solution hybridization, or quantitative reverse transcription PCR (qRT-PCR). Methods of carrying out these techniques are routine in the art. Additional methods of determining the level of mRNA in a sample are also well known to a person of ordinary skill in the art and such embodiments are within the purview of the invention.
[0108] Various techniques are known to a person of ordinary skill in the art to determine the level of PKC-ζ and/or PKC-.Math. protein in a sample. Non-limiting examples of such techniques include protein array analysis, Western blot analysis, enzyme-linked immunosorbent assay (ELISA), radio-immune assay (RIA), etc. Methods of carrying out these techniques are routine in the art. Additional methods of determining the level of PKC-ζ and/or PKC-.Math. protein in a sample are also known to a person of ordinary skill in the art and such embodiments are within the purview of the invention.
[0109] Similarly, various techniques are known to a person of ordinary skill in the art to determine the activity of PKC-ζ and/or PKC-.Math. protein in a sample. Methods of carrying out activity assays of PKC-ζ and/or PKC-.Math. protein are known in the art and are within the purview of the invention.
[0110] A reference value corresponding to the level of mRNA or protein for PKC-ζ and/or PKC-.Math. indicates the level of mRNA or protein for PKC-ζ and/or PKC-.Math. in the melanoma cells of a subject that is responsive or non-responsive to a melanoma therapy with an inhibitor of PKC-ζ and/or PKC-.Math.. As such, a reference value corresponding to the level of mRNA or protein for PKC-ζ and/or PKC-.Math. may be indicative of the subject being responsive or non-responsive to a melanoma therapy with an inhibitor of PKC-ζ and/or PKC-.Math..
[0111] To practice the methods of the subject invention described herein for identifying a subject as being responsive or non-responsive to a melanoma therapy with an inhibitor of PKC-ζ and/or PKC-.Math., a control sample can be one or more of the following:
[0112] a) melanocytes from an individual belonging to the same species as the subject and not having melanoma,
[0113] b) melanocytes from the subject prior to getting melanoma,
[0114] c) melanocytes from the subject not affected by melanoma,
[0115] d) melanoma cells from an individual known to be responsive to a melanoma therapy with an inhibitor of PKC-ζ and/or PKC-.Math., or
[0116] e) melanoma cells from an individual known to be non-responsive to a melanoma therapy with an inhibitor of PKC-ζ and/or PKC-.Math..
[0117] Additional examples of control samples that can be used in the invention can be designed by a person of ordinary skill in the art and such embodiments are within the purview of the invention.
[0118] In certain embodiments, the subject used in the invention is a mammal. Non-limiting examples of mammals include human, ape, canine, pig, bovine, rodent, or feline.
[0119] Once a subject is identified as being responsive to a melanoma treatment with an inhibitor of PKC-ζ and/or PKC-.Math. based on the methods of the subject invention, the steps of treating and/or managing melanoma include administering to the subject one or more inhibitors of PKC-ζ and/or PKC-.Math. described herein.
[0120] In preferred embodiments, a melanoma treatment with an inhibitor of PKC-ζ and/or PKC-.Math. is administered to the subject in combination with one or more additional melanoma therapies. Such additional melanoma therapies can be therapies that do not inhibit PKC-ζ and/or PKC-.Math., a surgery, a radiation therapy, an immunotherapy or a combination thereof.
[0121] In other preferred embodiments, two inhibitors of inhibitor of PKC-ζ and/or PKC-.Math. are administered to the subject in combination. In further preferred embodiments, the two inhibitors of PKC-ζ and/or PKC-.Math. that are administered to the subject in combination are administered in a way that each inhibitor is administered at a sub-therapeutic level.
[0122] Once a subject is identified as being non-responsive to a melanoma treatment with an inhibitor of PKC-ζ and/or PKC-.Math. based on the methods described herein, the step of treating and/or managing melanoma includes administering to the subject one or more melanoma therapies other than an inhibitor of PKC-ζ and/or PKC-.Math.. Examples of melanoma therapies other than an inhibitor of PKC-ζ and/or PKC-.Math. include a surgery, a radiation therapy, an immunotherapy or a combination thereof.
[0123] In specific preferred embodiments, the subject invention provides a novel atypical PKC inhibitor that is specific to PKC-ζ. In further preferred embodiments, the novel atypical PKC inhibitor specific to PKC-ζ is 8-hydroxy-1, 3,6-naphthalenetrisulfonic acid (ζ-Stat). Advantageously, the novel PKC-ζ specific inhibitor can decrease the levels of total and phosphorylated PKC-ζ without affecting PKC-.Math.. In preferred embodiments of the subject invention, the PKC-ζ specific inhibitor ζ-Stat inhibits cellular signaling pathways that are involved in endothelial-to-mesenchymal transition (EMT) of melanoma cells. Further advantageously, the PKC-ζ specific inhibitor 8-hydroxy-1, 3,6-naphthalenetrisulfonic acid (ζ-Stat) effectively suppresses PKC-ζ mediated EMT and induces apoptosis in melanoma cells.
[0124] All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.
[0125] Following are examples which illustrate procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.
EXAMPLES
Example 1: Materials
[0126] ACPD (Product #: R42691 1) was purchased from Sigma Aldrich (St. Louis, Mo.) and DNDA was obtained from the National Institute of Health-NIH (Bethesda, Md.). They were dissolved in sterile distilled water (vehicle) before use. ζ-Stat was obtained from the National Institute of Health-NIH (Bethesda, Md.). They were dissolved in sterile distilled water (vehicle) before use. PKC-.Math. (Catalog # 610175, 0.25 μg/mL) and Bcl-2 (Catalog # 610538, 0.5 μg/mL) antibodies were purchased from BD Biosciences (San Jose, Calif.). PKC-ζ (Catalog # sc-17781, 0.2 μg/mL), NF-kB p65 (Catalog # sc-372-G, 0.2 μg/mL) and Caspase-3 (Catalog # sc-7272, 0.4 μg/mL) antibodies was purchased from Santa Cruz Biotech (Santa Cruz, Calif.). The antibodies to phospho PKC-ζ (Thr 410) (Catalog # PAS-17837, 0.4 μg/mL), Phospho PKC-.Math. (Thr 555) (Catalog # 44-968G, 0.4 μg/mL), E-Cadherin (Catalog # 701134, 0.25 μg/mL) and Vimentin (Catalog # MA3-745, 0.25 μg/mL) antibodies were purchased from Thermo Fisher Scientific (Waltham, Mass.). PARP (Catalog # 9532, 0.15 μg/mL) and cleaved-PARP (Catalog # 9185, 0.15 μg/mL) antibodies were purchased from Cell Signaling (Danvers, Mass.). β-actin-peroxidase (Catalog # A3854, 0.1 μg/mL) antibody was purchased from Sigma (St. Louis, Mo.). CD44 (Catalog # ab51037, 0.2 μg/mL) and β-catenin (Catalog # ab16051, 0.2 μg/mL) antibodies were purchased from Abcam (Cambridge, UK). Enhanced chemiluminescence (Supersignal West Pico Chemiluminescent Substrate) solution (product #: 34080) was purchased from Pierce (Rockford, Ill.). Dulbecco's phosphate buffered saline without Mg2+ and Ca2+ (DPBS) (product #: D8537) and Trypsin-EDTA (Ethylenediaminetetraacetic acid) solution (product #: T4049) was purchased from Sigma Aldrich (St. Louis, Mo.). WST-1 reagent for cell proliferation, viability and cytotoxicity (catalog #: 11644807001) was purchased from Roche Diagnostics (Mannhelm, Germany).
Example 2: Database Preparation and Molecular Docking
[0127] Database preparation were performed using the National Cancer Institute/Developmental Therapeutics Program (NCl/DTP) and molecular docking was performed by selecting structural pockets in PKC-.Math. and PKC-ζ proteins which may have the potential of having interactions with small drug like molecules. The detailed procedure was performed as described [35].
Example 3: Cell Culture, Media, Cell Lines and Culture Conditions
[0128] PCS-200-013 normal melanocyte (derived from the epidermis-basal layer) and SK-MEL-2 metastasized melanoma cell lines (skin derived tissue from metastasis on skin of thigh of a 60 years old Caucasian male). MeWo, was derived from a skin metastasis to the lymph node of a 78 years old Caucasian male. All 3 cell lines were purchased from American Type Tissue Culture Collection (ATCC; Rockville, Md.). Both cell lines were cultured at 37° C. with 5% CO.sub.2.
[0129] Dermal cell basal medium and the growth kit for normal human melanocytes were also obtained from the ATCC. PCS-200-013 cells were seeded and grown as monolayers in 25 cm.sup.2 flasks in low serum (less than 1.0% FBS) conditions in the absence of cholera toxin and phorbol 12-myristate 13-acetate (PMA). The medium contained rh-Insulin (5 μg/mL), Ascorbic Acid (50 μg/mL), L-Glutamine (6 mM), Epinephrine (1.0 μM), Calcium Chloride (1.5 mM), Penicillin (5 μg/mL), Peptide Growth Factor and M8 supplement as proprietary formulations. SK-MEL-2 and MeWo cells were seeded and grown as monolayers in 25 cm.sup.2 flasks containing Eagle's minimum essential media-EMEM (90% v/v), fetal bovine serum-FBS (10% v/v) and Penicillin (5 μg/mL). Peptide Growth Factor and M8 supplement as proprietary formulations. SK-MEL-2 and MeWo cells were seeded and grown as monolayers in 25 cm.sup.2 or 75 cm.sup.2 flasks containing Eagle's minimum essential media-EMEM (90% v/v), fetal bovine serum-FBS (10% v/v) and Penicillin (5 μg/mL).
Example 4: Growth Inhibition Assays
[0130] For the growth inhibition assays, the PCS-200-013 cells or SK-MEL-2 cells were seeded at 40,000 cells/well into 6-well-plates and treated with the respective inhibitor. Cells in each well were counted after 24 h from the time of treating with the inhibitor at the desired concentration. Each inhibitor had triplicate plates which were counted at 24.sup.th, 48.sup.th and 72.sup.nd hours after initiating the inhibitor treatment. Inhibitors were added after 24 h of seeding point. The inhibitor concentrations were none (control), 50 nM, 100 nM, 200 nM, 500 nM and 1 μM. The cells were counted using a hemocytometer. The same procedure was followed for inhibitors ICA-1, ACPD, 3,4-diamino-2, 7-naphthalenedisulfonic acid DNDA and 8-hydroxy-1, 3,6-naphthalenetrisulfonic acid (ζ-Stat).
Example 5: Wound-Healing, Migration and Invasion Assays
[0131] For the wound-healing assay, melanoma cells were seeded into 35 mm dishes in 6-well-plates. After the cells had reached 100% confluency, the wound-healing assay was performed with a sterile pipette tip (1-10 μL pipette tip) by making a scratch through the confluent monolayer. Subsequently, the cells were washed with DPBS and fresh medium (EMEM) was added to each well and the desired amount of inhibitor was added in order to achieve the final concentrations of 50 nM, 100 nM, 200 nM, 500 nM and 1 μM against the control. Cells were cultured for 48 h. In 24 hours intervals, pictures of wound closure were taken and the percentage of wound closure was calculated in three randomly chosen fields.
[0132] For the transwell migration and invasion assays, approximately 2×10.sup.5 melanoma cells in serum-free medium were placed in the upper chamber of the insert with (invasion assay) or without (migration assay) Matrigel (BD Biosciences, San Jose, Calif.). Cells were treated with atypical PKC inhibitors to the upper chamber to achieve the final concentrations of 50 nM, 100 nM, 200 nM, 500 nM and 1 μM against the control. After 72 h incubation at 37° C., the cells remaining in the upper chamber or on the upper surface of the membrane were removed with a cotton swab. After staining with the suitable color reagent, the number of cells that adhered to the underside of the membrane was counted in three randomly chosen fields. Each experiment was performed in triplicate.
[0133] The same procedure was followed for inhibitors ICA-1, ACPD, 3,4-diamino-2,7-naphthalenedisulfonic acid DNDA and 8-hydroxy-1, 3,6-naphthalenetrisulfonic acid (ζ-Stat).
Example 6: Cell Fractionation, Immunoprecipitation and Western Blot Analysis
[0134] Approximately 2×10.sup.5 cells from PCS-200-013 and SK-MEL-2 cell lines were plated in T25 flasks until they become 70% confluent. Confluent cells were semi-synchronized by contact inhibition and serum starvation for 48 hrs. Subsequently, cells were incubated with serum and atypical inhibitors. The cells were removed at specific times and were placed in ice to terminate the incubation. Cells were washed at least two times with ice cold DPBS. Cells were then scrapped, re-suspended and sonicated in 500 μL of homogenization buffer (50 mM HEPES-pH 7.5), 150 mM NaCl, 0.1% Triton X, 1 mM EDTA (ethylenediaminetetraacetic acid), 2 mM EGTA [glycol-bis (2-aminoethylether)-N, N, N′,N′-tetraacetic acid], 0.1 mM orthovanadate, 1 mM NaF, 2 mM PMSF (phenylmethylsulphonylfluoride), 1 mM DTT (dithiothreitol) and 0.15 U/mL aprotinin. Cell suspensions was then sonicated and centrifuged at 100,000g for 30 minutes. The protein concentrations was determined using Bradford method. Subsequently, approximately 800 μg of proteins were immunoprecipitated with the antibody against desired protein. In some cases cell lysates were suspended with anti-HFS-1 antibody, rocking overnight at 4° C. Agarose beds were added after 24 hrs. Then the complex of cell lysate-anti HSF-1 antibody-agarose beads was rocked at 4° C. for 2 hrs. After washing the beads, approximately 60 of the sample loading buffer was added to dissolve all the protein on the beads. Proteins were then separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transblotted followed by immunoblotting with anti-PKC-ζ or anti-PKC-.Math. or anti-HSF-1 antibody or phosphospecific antibodies. Immunoreactive bands were visualized with enhanced chemiluminescence.
Example 7: PKC Activity Assay
[0135] PKC activity assay was conducted by monitoring the phosphorylation of myelin basic protein (MBP), a known substrate for PKCs. The detailed procedure was performed as described in Ling, D. S. F. et al. (2002) for PKC-.Math. and PKM-ζ [37].
[0136] PKC activity assay was performed by suspending 0.5 μg of PKC-ζ and PKC-.Math. separately in 200 μL of PKC kinase buffer to test the ability of PKC-ζ and PKC-.Math. of phosphorylating and immunoprecipitating HSF-1 of the SK-MEL-2 cells and PCS-200-013 cells. The PKC kinase buffer composition was 20 mM Tris-HCL (pH of 7.5), 6 mM Magnesium acetate, phosphotidylserine (5 μg, 20 μL of 10 mg/mL solution), ATP (0.0045 mg/mL) and also the desired inhibitors, wherever appropriate (ICA-1, ACPD, DNDA and ζ-Stat). The inhibitor was used in two different concentrations as 1 μM and 0.5 μM. The reaction was terminated after incubation for 5 minutes at 30° C. by addition of sample loading buffer. Samples were then separated by PAGE and immunoblotted against anti-PKC antibodies (either PKC-.Math. or PKC-ζ) and against phosphospecific HSF-1 antibodies. Samples were also probed against anti-HSF-1 antibody to determine that the protein immunoprecipitaed was HSF-1. The signal was visualized using enhanced chemiluminescence.
[0137] The same procedure was followed for inhibitors ICA-1, ACPD, 3,4-diamino-2,7-naphthalenedisulfonic acid (DNDA) and 8-hydroxy-1, 3,6-naphthalenetrisulfonic acid (ζ-Stat).
Example 8: Inhibitor Dose Response Curves
[0138] Dose curves (inhibitor concentrations vs cell population after 3 days) for ICA-1, ACPD, DND and -Stat were obtained for all 3 cell lines as follows. PCS-200-013, SK-MEL-2 and MeWo cells (4×10.sup.4) were cultured in 25 cm.sup.2 flasks and treated with either equal volume of sterile water (vehicle control). Cells were treated every 24 h during 3 day incubation period and cells were then lifted using Trypsin-EDTA solution (1.5 mL/flask) and neutralized with the equal volume of media. Subsequently, live cells were counted using the Scepter, an automated cell counter from Millipore (Billerica, Mass.). The cell growth inhibition for all inhibitors for the two malignant cell lines (SK-MEL-2 and MeWo) were quantified by counting the viable cells at 24 hour intervals. Cell counts obtained from Scepter was compared with the counts obtained from the Cellometer Vision from Nexcelom Bioscience (Lawrence, Mass.).
Example 9: Immunofluorescence
[0139] Approximately 75,000 cells were plated in a glass chamber slide. After 24 h post plating period, cells were washed 3 times with ice cold DPBS and were fixed with 1:1 Methanol to Acetone solution for 5 minutes at 20° C. Then, the cells were blocked in 8% normal goat serum for 45 minutes. This was followed by incubation with primary mouse antibody against PKC-.Math. and PKC-ζ accordingly for 90 minutes and subsequent incubation with anti-mouse secondary antibody for 30 minutes. Fluorescence Isothocyante fluorescing molecules were used for fluorescing PKC-.Math. and PKC-ζ proteins for 30 minutes. Cells were washed 3 times with DPBS prior to blocking with 8% normal horse serum for 45 minutes. Subsequently, cells were incubated with primary rabbit antibody against HSF-1 for 90 minutes and then incubated with anti-rabbit secondary antibody for 30 minutes. Texas Red fluorescing molecule was used to cause fluorescence of the HSF-1 protein for 30 minutes. Cells were washed three times with DPBS and were observed under Leitz Orthoplan scanning confocal microscope. Cell nuclei were visualized with mounting medium containing DAPI. Images were captured using a CCD camera with the Smart Capture Program. Photographs were taken with the same exposure of each setting for each experiment. To illustrate subcellular regions of protein co-localization, individual red and green stained images derived from the same field were merged and areas of protein co-localization appeared as yellow.
Example 10: NMR Experiments for Determining the Degradation of Inhibitors Over Time
[0140] .sup.1H NMR experiments were performed using 500 MHz Varian NMR spectrophotometer. Inhibitor concentration of 50 μM (ACPD, ICA-1, DNDA, and ζ-Stat) were initially maintained in the 5 mL growth media in a T25 flask. This solution was incubated at 37° C. and 5% CO.sub.2. Aliquots of 0.5 mL were taken out after 24 hours post incubation period and this was continued for 3 more days. These aliquots were analyzed for the .sup.1H NMR spectra of the respective drug to determine the changes in the concentrations compared to the reference spectra.
Example 11: WST-1 Assay for Cell Viability and Cytotoxicity
[0141] Approximately 4×10.sup.3 cells (PCS-200-013, SK-MEL-2 and MeWo) were cultured in a 96 well plate. After 24 h post plating time, fresh media were supplied (200 μL/well) and treated with either equal volume of sterile water (vehicle control) or with the half maximal inhibitory concentration (IC.sub.50) of ICA_1, ACPD, DNDA, or ζ-Stat. This IC.sub.50 was obtained based on the cell viability counts in the previous experiments. Additional doses of sterile water or inhibitor were supplied every 24 h during 3 day incubation period. At the end of 3 day treatment, media were removed and fresh media (100 μL) were added with 4-[3-(4-iodophenyl)-2-(4-nitropheny))-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) reagent (10 μL) to each well. The absorbance was measured at 450 nm for every 0.5 hour up to 3 hours using the Synergy HT microplate reader from Biotek (Winooski, Vt.).
Example 12: Statistical Analysis
[0142] All data are presented as mean±SD. Statistical analysis was performed with one or two-way ANOVA followed by Tukey HSD test as multiple comparisons tests using the “VassarStats” web tool for statistical analysis developed by Dr. Richard Lowry, PhD. P-value≦0.05 or ≦0.01 was considered to indicate statistical significance.
Example 13: Specific Binding of ACPD and DNDA to aPKCs
[0143] To establish the therapeutic potential of atypical PKCs, ACPD (
Example 14: Specificities of ACPD and DNDA Based on Activity Assays
[0144] Determination of specific activity of inhibitors was essential since 84% similarity is observed in the primary structures of the PKC-.Math. and PKC-ζ catalytic domains. Specificity of ACPD was previously reported as inhibiting both PKC-.Math., approximately 40% at 10 1 M, and PKC-ζ about 60% at 10 without affecting other PKC isoforms [24]. Additionally, ACPD does not inhibit other kinases such as MAPK, Akt2, FGFR 1/2/3/4, mTOR, GSK3B, IRAK1/4, JAK 1/2, MEK1, ERK1/2, JNK 1/2, PKA, Src, ROCK2 and PI3K [25, 26].
[0145] The activity of DNDA (
Example 15: Dose Curves for PKC-.Math. and PKC-ζ
[0146] Cell growth of malignant melanoma cells was reduced by applying PKC-.Math. and PKC-inhibitors (
TABLE-US-00001 TABLE 1 Growth inhibition by ICA-1. ICA-1 % Inhibition Concentration Day 1 Day 2 Day 3 0 (Control) 0 0 0 50 nM 4 8 7 100 nM 8 8 11 200 nM 11 20 20 500 nM 19 27 25 1 μM 25 30 34
TABLE-US-00002 TABLE 2 Growth inhibition using ACPD. ACPD % Inhibition Concentration Day 1 Day 2 Day 3 0 (Control) 0 0 0 50 nM 1 4 5 100 nM 11 10 9 200 nM 19 23 20 500 nM 26 30 26 1 μM 36 35 37
Example 16: Inhibitor Dose Response Curves
[0147] Dose curves for ACPD and DNDA were obtained to investigate the effect on cell proliferation of normal and malignant cell lines over a wide range of concentrations. ACPD and DNDA did not show a significant effect on PCS-200-013 (
Example 17: WST-1 Assay for Cell Viability and Cytotoxicity
[0148] WST-1 assay was performed to determine the in-vitro cytotoxicity of ACPD and DNDA on normal and malignant cell lines. Measured absorbance at 450 nm is directly proportional to the number of cells present. Cells produce a water soluble formazan with WST-1 as a result of their mitochondrial dehydrogenase activity. WST-1 assay is preferred over 3-(4,S-Dimethyl-2-thiazolyl)-2,5-diphenyl-2Htetrazolium bromide (MTT) test since MTT needs to add acidic isopropanol to dissolve formazan which gives an additional toxicity to cells [30]. Both inhibitors did not show a significant effect on normal cells (
Example 18: Wound Healing Assay for ACPD and ICA-1
[0149] The results of a wound healing assay are provided in
Example 19: Wound Healing Assay for ACPD and DNDA
[0150] A wound healing assay was performed to investigate the effect of ACPD and DNDA on malignant melanoma cell migration in vitro. Wound healing assay is commonly used to investigate in vitro migration of cancer cells [31-33]. For the wound-healing assay, melanoma cells were seeded into 35 mm dishes in 6-well-plates. After the cells had reached 100% confluency, the wound-healing assay was performed with a sterile pipette tip (1-10 μL pipette tip) by making a scratch through the confluent monolayer. Subsequently, the cells were washed with DPBS and fresh medium (EMEM) was added to each well and the desired amount of inhibitor was added. Each well was subsequently incubated with sterile water or desired amount of ICA-1, ACPD, DNDA, or ζ-Stat to achieve the final concentration of the half maximal inhibitory concentration (IC50) for each inhibitor. Plates were incubated at 37° C. and 5% CO.sub.2 and photographs of wound closure were taken utilizing a Motic AE3 lE microscope (40× magnification). The percentage of wound closure was calculated after 3 or 4 days.
[0151] In one set of experiments, cells were cultured for 48 h. In 24 hours intervals, pictures of wound closure were taken and the percentage of wound closure was calculated in three randomly chosen fields.
Example 20: BME Invasion Assay
[0152] This invasion assay was performed to investigate the effect of ACPD and DNDA on malignant melanoma cell invasion in vitro. Even though it is similar to the Boyden chamber assay, it avoids scraping off the Matrigel and staining to assess migrated number of cells through filter. Hence, the method carries less human error compared to conventional Boyden chamber assay. Migrated cells were stained with a fluorescent marker, Calcein-AM. Live cells cleave the ester (AM) of the molecule in order to produce fluorescence. Thus, the amount of fluorescence accumulated in the bottom chamber is proportional to the number of invaded cells. The relative fluorescent units (excitation at 485 and emission at 528 nm) after 2 h exposure were reported for inhibitor treatments for both SK-MEL-2 and MeWo cell lines compared to controls (
Example 21: Effect of Inhibitors on aPKC Levels in Malignant Melanoma
[0153] PKC-.Math. expression was examined in western blot analysis for ICA-1 and ACPD treated samples. ICA-1 treated samples showed a significant decrease of PKC-.Math. level upon increasing the ICA-1 concentration (
Example 22: Effects of ACPD and DNDA on aPKC and Phospho-aPKC Expression
[0154] Western blots (WB) were performed to investigate the effect of ACPD and DNDA on the expression of aPKCs on malignant melanoma. As shown in
[0155] As shown in
Example 23: Effect of ACPD and DNDA on Apoptosis of Malignant Melanoma
[0156] Since both inhibitors effectively inhibit melanoma cell proliferation, the inventors tested the potential of the inhibitors on inducing the apoptosis. As shown in
Example 24: Effect of Inhibitors on EMT
[0157] As shown in
Example 25: Association of PKC-z and Vimentin
[0158] PKC-.Math. and PKC-ζ were immunoprecipitated (IP) separately and western blot experiments were conducted independently for E-cadherin, CD44, vimentin and NF-κB p65. PKC-ζ IP samples did not show any association with any mentioned proteins. Only vimentin western blot showed a band for PKC-.Math. IP samples (
Example 26: Specific Binding of ζStat to PKC-ζ
[0159] The chemical structure of -Stat and the molecular docking (MD) of ζ-Stat on PKC-ζ are shown in
Example 27: Effects of ζ-Stat on Malignant Cells
[0160] To test the effects of ζ-Stat on malignant cells approximately 4×10.sup.4 of cells were cultured in 25 cm2 flasks and treated with either equal volume of sterile water (control) or ζ-Stat (0.1-10 μM). Additional doses of sterile water or inhibitor were supplied every 24 h during 3 day incubation period. Subsequently, cells were lifted and counted. Cells were quantified by counting the viable cells at 24 hour intervals. The results showed the effect of -Stat on SK-MEL-2 (
Example 28: Effects of ζ-Stat from WST-1 Assay for Cell Viability and Cytotoxicity
[0161] Cell proliferation was measured using WST-1 assay for SK-MEL-2 (
Example 29: Effects of ζ-Stat on Melanoma Cell Migration Using Wound Healing Assay
[0162] Microscopic photographs of scratches on cells at the beginning (day 0) were compared with the photographs taken after 3 or 4 days the effects of inhibitors are shown for SK-MEL-2 (
Example 30: Effects of Inhibitors on Expression of aPKC as Determined by Western Blots
[0163] The change of levels of phosphorylated PKC-.Math., total PKC-.Math. (
[0164] Based on these results, ζ-Stat can effectively reduce the malignancy of melanoma cell lines in vitro. Molecular docking confirmed that ζ-Stat specifically binds to PKC-ζ and results of kinase activity assay show more than 50% of inhibition of PKM-ζ activity (a homologous of PKC-ζ found in human brain) but it does not effect on PKC-.Math.. PKC-ζ acts as a pro-growth protein in many human cancer cells/tissues. Therefore, effective inhibition can lead to cure malignant tumors based on specific markers. According to the WST-1 assay, ζ-Stat did not show toxicity to malignant melanoma cells at the tested concentration (2.5 μM) which had only 20-25% inhibition of cell viability according to the cell counts on viability. But 5 μM and 10 μM concentrations produced an inhibition close to 50% hence future tests will be performed using 7-7.5 μM of ζ-Stat. At the tested concentration, the protein levels of total PKC-.Math. and phosphorylated PKC-.Math. levels did not change significantly but total PKC-ζ level was reduced by 42% and the pPKC-.Math. level reduced by 37%. Additionally, we found that ζ-Stat plays a role in altering the levels of proteins which are used as epithelial to mesenchymal transition (EMT). We also found that treatment of ζ-Stat (C-50) induces the apoptosis of malignant melanoma cells.
[0165] It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims. In addition, any elements or limitations of any invention or embodiment thereof disclosed herein can be combined with any and/or all other elements or limitations (individually or in any combination) or any other invention or embodiment thereof disclosed herein, and all such combinations are contemplated within the scope of the invention without limitation thereto.
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