Methods and compositions for inhibiting the proliferation of prostate cancer cells

TECHNICAL FIELD

[0002] This invention relates to prostate cancer, and more particularly to methods and compositions for inhibiting the proliferation of prostate cancer cells.

BACKGROUND

[0003] The prostate gland is located between the bladder and the rectum and wraps around the urethra. The prostate is composed of glandular tissue that produces a milky fluid and smooth muscles that contract during sex and squeeze this fluid into the urethra where it mixes with other fluid and sperm to form semen. The prostate gland converts testosterone to a more powerful male hormone, dihydrotestosterone, which affects the size of the gland and plays an important role in prostate cancer.

[0004] Prostate cancer is a malignant tumor that arises in the prostate gland and can eventually spread through the blood and lymph fluid to other organs, bones, and tissues. Prostate cancer is the most commonly diagnosed cancer in the U.S., and it is the second leading cause of cancer death in American men after non-melanoma skin cancer. Although prostate cancer is just as common in Japan as in the United States, death rates from prostate cancer are significantly lower in Japan. It is unlikely that these differences are all genetic, because Japanese men who migrate to the United States die of prostate cancer with increasing frequency as a function of the number of years they reside in the United States. It is possible that this paradox could be explained, at least in part, by dietary factors.

[0005] Benign prostatic hyperplasia (BPH) is a benign enlargement of the prostate gland caused by the growth of both glandular and stromal tissues. Because the prostate enlargement in BPH is affected by testosterone, many men are concerned that it may be related to prostate cancer. A ten-year study, however, found no higher risk for prostate cancer in men with or that have experienced BPH. BPH develops in the inner zone of the prostate (i.e., predominantly stromal cells), while cancer tends to develop in the outer area (i.e., epidermal cells).

SUMMARY

[0006] It is reported herein that the nuclear localization of the androgen receptor was inhibited by silymarin. Accordingly, the invention provides for methods of monitoring the proliferation of cultured prostate cancer cells in the presence of silymarin, methods of treating an individual with prostate cancer or at risk of developing prostate cancer, and methods of reducing the risk of recurrence of prostate cancer in an individual who had previously been treated for prostate cancer. Methods of the invention further include treating an individual with benign prostatic hyperplasia (BPH) as well as methods of screening for compounds that inhibit the proliferation of prostate cancer cells. The invention provides for compositions and articles of manufacture containing silymarin in particular formulations, or silymarin with a second compound that also exerts an effect on the androgen receptor.

[0007] In one aspect, the invention provides methods of monitoring the proliferation of cultured prostate cancer cells in the presence of silymarin or a component thereof. Such a method includes contacting cultured prostate cancer cells with silymarin or a component thereof and monitoring the amount of nuclear localization of an androgen receptor. Generally, a decrease in androgen receptor nuclear localization indicates an inhibitory effect by silymarin or a component thereof on the proliferation of the prostate cancer cells. Representative prostate cancer cell lines include LNCaP cells or LAPC-4 cells. The component of silymarin can be silibin.

[0008] In another aspect, the invention provides methods of treating an individual with prostate cancer or at risk of developing prostate cancer. Methods of treating an individual with prostate cancer or at risk of developing prostate cancer include identifying an individual with prostate cancer or at risk of developing prostate cancer and administering a dose of silymarin or a component thereof to the individual that is effective to inhibit the nuclear localization of an androgen receptor. The method further can include the step of monitoring the amount of nuclear localization of the androgen receptor in the individual. Decreasing androgen receptor nuclear localization inhibits the proliferation of prostate cancer cells, thereby treating the individual. For example, silymarin or a component thereof can be administered to a human, and in an amount of from about 50 mg/kg to about 500 mg/kg. Silymarin or a component thereof can be administered orally, transdermally, intravenously, intraperitoneally, or using an implant.

[0009] In still another aspect, the invention provides for methods of reducing the risk of recurrence of prostate cancer in an individual, wherein the individual previously had been treated for prostate cancer. Such a method includes the step of administering a dose of silymarin or a component thereof to the individual that is effective to inhibit the nuclear localization of an androgen receptor. The method can further include the step of monitoring the nuclear localization of the androgen receptor in the individual. Generally, inhibiting androgen receptor nuclear localization inhibits the proliferation of prostate cancer cells, thereby reducing the risk of recurrence of prostate cancer in the individual. The individual may have previously undergone a radical prostectomy.

[0010] In yet another aspect, the invention provides methods of treating an individual with benign prostatic hyperplasia (BPH). This method includes identifying an individual with BPH and administering a dose of silymarin or a component thereof to the individual that is effective to inhibit the nuclear localization of an androgen receptor. The method further can include monitoring the nuclear localization of the androgen receptor in the individual. Inhibiting the nuclear localization of the androgen receptor thereby treating the BPH in the individual.

[0011] The invention additionally provides methods of screening for compounds that inhibit the proliferation of prostate cancer cells, including contacting prostate cancer cells with a compound, and determining the amount of nuclear localization of an androgen receptor. The method also can include monitoring the amount of nuclear localization of the androgen receptor in the prostate cancer cells. Decreased androgen receptor nuclear localization in the prostate cancer cells compared to prostate cancer cells not contacted with the compound indicates a compound that inhibits the proliferation of prostate cancer cells. Prostate cancer cells such as LNCaP cells or LAPC-4 cells can be used in this method.

[0012] Further, the invention provides compositions that include silymarin or a component thereof, one or more compounds that has a particular mechanism of action (i.e., inhibiting expression of a gene encoding an androgen receptor, inhibiting the nuclear localization of an androgen receptor, and inhibiting the transactivating ability of an androgen receptor), and a pharmaceutically acceptable carrier. Representative examples of compounds having such particular mechanisms of action include quercetin, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), perillyl alcohol (POH) or a derivative thereof, resveratrol, flufenamic acid, tea polyphenols, and anti-androgen compounds. It is a feature of the invention to provide such a composition in the form of an article of manufacture (e.g., a kit). Such an article of manufacture can include packaging material comprises instructions for using the composition to inhibit the nuclear localization of an androgen receptor in an individual.

[0013] In another aspect of the invention, there are provided compositions that include silymarin or a component thereof and that are formulated for transdermal delivery to the prostate of an individual. Delivery to the prostate inhibits androgen receptor nuclear localization. In addition, the invention provides compositions that include silymarin or a component thereof and that are formulated for implantation near the prostate of an individual, wherein the implantation near the prostate inhibits the nuclear localization of an androgen receptor.

[0014] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0015] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the drawings and detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

[0016]
FIG. 1 shows the effects of silymarin and silibin on androgen-regulated cell growth in LNCaP cells. *p<0.05, when compared with the group treated with Mib alone.

[0017]
FIG. 2 shows the effects of silymarin and silibin on the expression of androgen-regulated proteins PSA (FIG. 2A), and hk2 (FIG. 2B). *p<0.05, when compared with the group treated with Mib alone.

[0018]
FIG. 3 shows the effects of silymarin and silibin on the 6 Kb PSA promoter's androgenic inducibility (FIG. 3A) and hk2 ARE-mediated transcriptional activity (FIG. 3B) in LNCaP cells. *p<0.05, when compared with the group treated with Mib alone. A percentage of activities is expressed on the top of the histograms using the PSA promoter or hK2-ARE construct treated with Mib as a reference (expressed as 100).

[0019]
FIG. 4 shows the effects of silymarin and silibin on whole cell androgen receptor levels (FIG. 4A), the transcription activity of the androgen receptor promoter (FIG. 4B), and the ligand-binding activity of the androgen receptor (FIG. 4C). * p<0.05, when compared with the group treated with Mib alone.

[0020]
FIG. 5A shows a bar graph demonstrating the whole cell androgen receptor levels in the presence of silymarin determined by immunohistochemical staining. FIG. 5B shows the ratio of the nuclear androgen receptor levels over the whole cell androgen receptor levels determined by immunohistochemical staining. *p<0.05, when compared with the group treated with Mib alone.

[0021] Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

[0022] It is reported herein that the transactivating activity of the androgen receptor was inhibited by silymarin or a component thereof. Accordingly, the invention provides for methods of monitoring the proliferation of cultured prostate cancer cells in the presence of silymarin or a component thereof, methods of treating an individual with prostate cancer or at risk of developing prostate cancer, and methods of reducing the risk of recurrence of prostate cancer in an individual who had previously been treated for prostate cancer. The invention further includes methods treating an individual with benign pro static hyperplasia (BPH) as well as methods of screening for compounds that inhibit the proliferation of prostate cancer cells. The invention provides for compositions and articles of manufacture containing silymarin or a component thereof in particular formulations, or silymarin or a component thereof with a second compound that also exerts an effect on the androgen receptor.

[0023] It was shown herein that silymarin or a component thereof inhibited androgen-stimulated induction of both prostate-specific antigen (PSA) and hK2. The nuclear localization of the androgen receptor was diminished by silymarin or a component thereof. The invention provides a novel aspect of silymarin in that silymarin or a component thereof can attenuate androgen receptor-mediated transactivation of prostate cancer-specific genes in androgen-responsive prostate cancer cells. Thus, the invention provides for methods of preventing or treating prostate cancer using silymarin or a component thereof.

[0024] The Androgen Receptor and Prostate Cancer

[0025] Androgens play an important role in the proliferation, differentiation, maintenance, and function of the prostate. The androgen receptor is the essential mediator for androgen action and is a ligand-dependent transcription factor belonging to the nuclear steroid hormone receptor superfamily. Androgens can enhance androgen receptor protein levels by increasing the half-life, as well as by stimulating the phosphorylation of the androgen receptor. Phosphorylation may affect numerous characteristics of nuclear receptors including ligand binding, nuclear translocation, dimerization, DNA binding, and protein-protein interactions.

[0026] Evidence shows that androgens are also involved in the development and progression of prostate cancer. Therefore, the androgen receptor also plays a critical role in the development of prostate cancer, in part due to overstimulation of the receptor by androgens. Prostate cancer also has been attributed to altered transactivation activities of the receptor or to mutations in the androgen receptor that, for example, enable the receptor to respond to non-androgen steroids. The androgen receptor can be expressed in all stages of prostate cancer, and at least one-third of advanced prostate cancers contain amplified androgen receptor genes.

[0027] The utilization of androgen deprivation as a treatment for advanced prostate cancer was first demonstrated in 1941 and has become a standard treatment. Based on the morbidity associated with ablation of the adrenal glands, castration alone was the gold standard until the 1980s, when anti-androgen agents, including cyproterone acetate, megestrol acetate, and flutamide, were developed to compete with androgen for binding to the androgen receptor. Many new classes of drugs that interfere with androgen production and function have been identified.

[0028] In spite of the apparent regression of tumors by hormone therapy, however, prostate cancer often recurs within 3 years and becomes hormone refractory with a potentially fatal outcome. Many molecular mechanisms have been postulated to be responsible for the development of recurrent hormone-refractory tumors with most involving alterations in the function of the androgen receptor and its complex signaling pathways. The androgen receptor can be activated by a number of growth factors or cytokines in the absence of androgens or by low levels of androgens or other non-androgenic steroid hormones after hormone therapy. That the majority of hormone-refractory cancers still express the androgen-responsive prostate-specific antigen PSA is a protein secreted by the epithelial cells of the prostate gland, including prostate cancer cells. An abnormally high level of PSA is indicative of abnormal prostate cells. (PSA) gene indicates that the androgen receptor signaling pathway is functional.

[0029] Nucleic acid sequences encoding androgen receptors have been cloned and sequenced from numerous organisms. Representative organisms and GenBank accession numbers for androgen receptor sequences therefrom include the following: frog (Xenopus laevis, U67129), mouse (Mus musculus, 109558), rat (Rattus norvegicus, 292896), human (Homo sapiens, 105325), rabbit (Oryctolagus cuniculus, 577829), cow (Bos taurus, Z75313, Z75314, Z75315), canary (Serinus canaria, 414734), and whiptail lizard (Cnemidophous uniparens, 1195596). Additionally, Cancer Genetics Web (www.cancer-genetics.org) contains database entries for wild-type and mutant androgen receptor sequences.

[0030] Silymarin

[0031] Flavonolignan extracts from the fruits of the milk thistle (Silybum marianum (L.) Gaertn., syn. Carduus marianus L.) contain silymarin, an isomeric mixture of silibin (also known as silibinin, silybin or silybinin), isosilibinin, silicristin and silidianin. See Wagner et al. (Arzneim-Forsch Drug Res., 1968, 18:688-96) for a description of the chemistry of silymarin. Silibin exhibits the primary biological activity, while the three remaining compounds in silymarin have less physiological activity: isosilibinin occurs in very small amounts; silidianin is extensively metabolized; and silicristin is poorly absorbed by the gastrointestinal tract. Numerous studies on silymarin and milk thistle extracts have shown them to be virtually without side effects when used in therapeutic doses (see, for example, Hobbs, Milk Thistle. The Liver Herb, 2nd Ed., Botanica Press, Capitola, Calif., 1992, and references therein).

[0032] Silymarin has been used orally for the treatment of toxic liver damage (induced by alcohol, drugs, or environmental toxins) and for supportive therapy in chronic inflammatory liver diseases and liver cirrhosis. Silymarin and its main isomer, silibin, have been shown to possess antioxidant properties, thus reducing or preventing lipid peroxidation and membrane destruction in cells. In addition, protein biosynthesis and cell regeneration are accelerated in the liver in the presence of silymarin, leading to restoration of liver functions. Silymarin exhibits anti-inflammatory activity, which may be a result of an inhibition by silymarin on leukotriene production. Specifically, silymarin is also noted for helping relieve biliary tract inflammation, and can further protect the liver from acetaminophen overdose.

[0033] Silymarin is slightly soluble in water and, therefore, is found in nuclear membranes, cell membranes, and the cytoplasm. Silymarin is primarily excreted in bile, although a relatively small amount is excreted by the kidneys. Silymarin has an affinity for the liver, partially due to its enterohepatic circulation. As silymarin is absorbed by the intestinal tract, it passes directly into the liver via normal portal circulation where it is taken up by hepatocytes and excreted into bile. The silymarin contained in bile is then reabsorbed by the intestines. Silymarin then is transported from the intestines to the liver to the gall bladder and back into the intestines. This enterohepatic circulation is thought to be the reason silymarin does not express its antioxidant properties on other organs such as the lungs, kidneys or spleen.

[0034] α-amanitin and phalloidin, the most potent toxins from the mushroom Amanita phalloides, circulate in an individual via an enterohepatic pathway. α-amanitin effectively shuts down the liver by binding to Polymerase A and preventing protein synthesis. Because of the enterohepatic circulation of α-amanitin, the liver is barraged by the poison and crippled. If the dose of α-amanitin is high enough (about 50+ grams of fungus), the individual will die. Silymarin is antagonistic to α-amanitin and decreases the toxic effects, for example, by changing the membrane integrity of hepatocytes, by making penetration of the cells by the poison more difficult, by competitive inhibition of receptors at the cell membrane, and/or by blocking the binding of α-aminitin to Polymerase A. Silymarin is also antagonistic to lanthanide poisoning, carbon tetrachloride, galactosamine, the hepatotoxic virus FU3 and other toxins.

[0035] Methods of Monitoring and Inhibiting the Proliferation of Prostate Cancer Cells

[0036] The invention provides for methods of monitoring the proliferation of prostate cancer cells. According to the methods of the invention, the proliferation of prostate cancer cells can be monitored by contacting those cells with silymarin or a component thereof and then determining the nuclear localization of the androgen receptor using conventional methods (e.g., methods described herein). A decrease in the nuclear localization is indicative of an inhibitory effect by silymarin or a component thereof on the proliferation of the prostate cancer cells. Proliferation of prostate cancer cells as used herein refers to an increase in the number of prostate cancer cells (in vitro or in vivo) over a given period of time (e.g., hours, days, weeks, or months). It is noted that the number of prostate cancer cells is not static and reflects both the number of cells undergoing cell division and the number of cells dying (e.g., by apoptosis). An inhibition of the proliferation of prostate cancer cells can be defined as a decrease in the rate of increase in prostate cancer cell number, a complete loss of prostate cancer cells, or any variation therebetween. With respect to tumors, a decrease in the size of a tumor can be an indication of an inhibition of proliferation.

[0037] Prostate cancer cells that can be maintained in culture and are useful in the invention include without limitation LNCaP cells and LAPC-4 cells. The LNCaP cell line is an established androgen-responsive prostate cancer cell line obtained from a lymph node metastasis of a prostate cancer patient. LNCaP cells express the androgen receptor and a number of androgen-inducible genes such as PSA, human glandular kallikrein (hK2), NKX3.1 and ornithine decarboxylase (ODC). The gene encoding the androgen receptor in the LNCaP cell line contains a mutation in its ligand binding domain, but otherwise is functional. LAPC-4 cells, another androgen responsive prostate cancer cell line suitable for use in the invention, expresses a wild-type androgen receptor. LAPC-4 cells additionally express PSA and hK2, which are up-regulated in the LAPC-4 cells by androgens. Other prostate cancer cell lines are available and include PC-3 and DU145.

[0038] The invention further provides for methods of treating an individual with prostate cancer or at risk of developing prostate cancer. An individual is first identified as having prostate cancer or being at risk for developing prostate cancer and then administered an effective dose of silymarin or a component thereof. The nuclear localization of the androgen receptor can be monitored in the individual to evaluate the effects of silymarin or a component thereof on prostate cancer cells. Generally, an inhibition of the nuclear localization of the androgen receptor by silymarin or a component thereof inhibits the proliferation of prostate cancer cells, thereby treating the individual.

[0039] Prostate cancer cells can be identified using several criteria. Prostate cancer cells in culture (e.g., LNCaP cells) can be characterized by the response of such cells to androgens or androgenic agonists or antagonists. Molecular markers, such as increased or decreased expression of androgen-regulated genes or genes involved in prostate cancer (e.g., PSA, hk2, c-jun, ODC, and NKX3.1) also can be used to characterize prostate cancer cells in culture. Prostate cancer in vivo can be identified by a digital rectal examination of a patient, or by imaging or scanning techniques (e.g., magnetic resonance imaging (MRI), or prostascint scans). In addition, the degree of cellular differentiation can be evaluated in prostate cancer cells from an individual, typically removed via a biopsy of prostate tissue, using a Gleason score. Further, there are several commercially available diagnostic tests for PSA and PSA-II (e.g., Roche Diagnostics Inc., Indianapolis, Ind.) to screen individuals for prostate cancer and to monitor individuals undergoing treatment for prostate cancer. Prostate cancer can be staged, for example, using a Partin Table and/or a Partin II Table (see Partin et al., 1994, Urology, 43:649-59 and http://www.theraseed.com/gloss.html for more information).

[0040] For the purpose of this invention, silymarin or a component thereof can be administered orally, transdermally, intravenously, intraperitoneally, or by implantation. The route of administration typically depends on a variety of factors, such as treatment environment and therapeutic goals. Administration of silymarin or a component thereof can be on a continuous or an intermittent basis. In addition, preparations for administration of silymarin or a component thereof can be suitably formulated to give controlled release of the compound. Preparations for intravenous and intraperitoneal administration can include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include, without limitation, propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters. Aqueous carriers include, without limitation, water, as well as alcohol, saline, and buffered solutions. Other additives such as, for example, antimicrobials, anti-oxidants, chelating agents, inert gases, steroids, anti-inflammatory agents, immunosuppressants, vasodilators, vasoconstrictors, and the like may also be present.

[0041] Tablets or capsules for oral administration can be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Tablets can be coated by methods known in the art. Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspension, or they can be presented as a dry product for constitution with saline or other suitable liquid vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl- or propyl-p-hydroxybenzoates or sorbic acid). The preparations can also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.

[0042] Preparations for transdermal administration are known in the art. Such transdermal preparations can be in the form of a scrotum patch or a patch for application on the back, abdomen, thighs or buttocks. A transdermal patch typically includes a soft flexible backing (e.g., polyester or polyester/ethylene-vinyl acetate copolymer), a reservoir (in some cases, the compound or composition, e.g., silymarin or a component thereof, can be deposited as a film on the ethylene-vinyl acetate copolymer or can be combined with, for example, alcohol and a gelling agent such as hydroxypropyl cellulose), and an adhesive backing made out of, for example, polyisobutylene and colloidal silicon dioxide (usually with a removable liner (e.g., silicone-coated polyester, or fluorocarbon diacrylate) to protect the adhesive until the patch is applied). A transdermal patch also can contain a formulation (e.g., polyisobutylene adhesive) to control the rate of release of the compound or composition.

[0043] Implantable devices are known in the art and can be in the form of a pellet or a seed containing or coated with a compound or composition, e.g., silymarin or a component thereof. A pellet or seed can be a metal alloy (e.g., cobalt, or palladium) or an inert plastic or other substance. A device for implantation in or near the prostate can be delivered using a delivery catheter (similar to brachytherapy) and can be deposited in or near the prostate transperineally, transrectally, or transurethrally. A transrectal ultrasound can be used in conjunction with implantation to visualize and image the prostate and the positioning of the implantable device.

[0044] According to the invention, an effective dose of silymarin or a component thereof is an amount that inhibits the nuclear localization of the androgen receptor, thereby inhibiting the proliferation of prostate cancer cells. Inhibition of the nuclear localization of the androgen receptor and the subsequent inhibition of the proliferation of prostate cancer cells can be determined using methods and assays described herein. It is anticipated that an effective dose of silymarin or a component thereof is from about 50 mg of silymarin or a component thereof per kg weight of the individual (mg/kg) to about 500 mg/(g. Toxicity and therapeutic efficacy of different doses of silymarin or a component thereof can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio of LD50/ED50. Doses of silymarin or a component thereof that exhibit high therapeutic indeces are preferred. An effective dose of silymarin or a component thereof can be delivered in a single dose or as multiple doses over a period of time.

[0045] The ability of the androgen receptor to translocate to the nucleus can be evaluated in the presence and absence of a compound to determine if the compound inhibits the nuclear localization of the androgen receptor. Nuclei are typically isolated using an appropriate gradient such as a sucrose gradient, a percol gradient, or the like. The nuclei can be lysed (for example, by exposure to sonication, or ultrasound waves) and androgen receptor protein can be detected using routine methods such as Western blotting. Nuclear translocation also can be examined using, for example, immunocytochemistry to identify androgen receptor protein in the nucleus and/or outside of the nucleus.

[0046] In addition, the invention provides methods of reducing the risk of recurrence of prostate cancer in an individual that previously had undergone treatment for prostate cancer. Such methods include administering an effective dose of silymarin or a component thereof to the individual such that the nuclear localization of the androgen receptor is inhibited. Inhibiting the nuclear localization of the androgen receptor inhibits the proliferation, and therefore the recurrence, of prostate cancer cells. Treatments for prostate cancer that an individual might undergo include hormone therapy, chemotherapy, radiation therapy and, oftentimes, a prostatectomy, in which part of all of the prostate gland is removed. A radical prostatectomy includes removal of the entire prostate as well as the seminal vesicles. Due to a high incidence of prostate cancer recurring, even following such treatments (including a radical prostatectomy), methods of the invention provide for administration of silymarin or a component thereof during or following such treatments. Administration of silymarin or a component thereof may be particularly useful following a radical prostatectomy.

[0047] The invention additionally provides for a method of treating an individual with benign prostatic hyperplasia (BPH). Individuals with BPH may present with prostatitis and/or difficulty urinating, and an enlarged prostate due to BPH is typically palpable during a digital rectal exam. Methods of the invention include identifying an individual with BPH, and administering a dose of silymarin or a component thereof or a derivative thereof to said individual effective to inhibit the nuclear localization of an androgen receptor. Such an inhibition of the androgen receptor's nuclear localizing ability reduces the androgen receptor-mediated growth response and thereby treats the individual with BPH.

[0048] Methods of Screening Compounds

[0049] The invention provides for methods of screening for compounds that inhibit the proliferation of prostate cancer cells by decreasing the nuclear localization of the androgen receptor. Screening methods are one of the fundamental tools used in molecular biology for rapid and efficient evaluation of compounds. Screening methods of the invention include contacting prostate cancer cells with a compound under conditions and for a time sufficient to allow the compound to enter the cell, and determining the nuclear localization of the androgen receptor. Generally, decreased nuclear localization of the androgen receptor in cells compared to cells not contacted with the compound indicates a compound that inhibits the proliferation of prostate cancer cells. Such compounds can be evaluated using prostate cancer cells in culture, such as LNCaP or LAPC-4 cells, or can be evaluated using a cell-free system.

[0050] Methods of determining the nuclear localization of the androgen receptor are described above. Expression of a gene encoding an androgen receptor in prostate cancer cells can be examined in the presence and absence of a compound using Northern blot analysis (to evaluate transcription) and/or Western blot analysis (to evaluate translation). Techniques to isolate RNAs and proteins from cells as well as methods of separation (e.g., electrophoretically) are well known and routine in the art. Androgen receptor mRNA can be detected by hybridization with a labeled oligonucleotide probe that is complementary to a portion of the androgen receptor transcript. Androgen receptor proteins can be detected by contacting proteins from a cell with a labeled agent that selectively binds to the androgen receptor protein. Conditions for allowing and detecting hybridization of nucleic acids or binding of antibodies to proteins are well known in the art. Antibodies that have binding affinity to androgen receptor proteins are commercially available (e.g., from Research Diagnostics Inc. (Flanders, N.J.) and Alpha Diagnostic International (San Antonio, Tex.)). The term “label”, with regard to an oligonucleotide probe or an antibody is intended to encompass direct labeling of the oligonucleotide or antibody by coupling a detectable substance to the oligonucleotide or antibody, as well as indirect labeling of the oligonucleotide or antibody by reactivity with a detectable substance. Examples of labels and detectable substances are well known in the art. Additional methods to detect androgen receptor mRNA (e.g., RT-PCR or dot blots) or protein (e.g., immunoassays or chromatography) are well known and also practiced routinely in the art.

[0051] The transactivation ability of the androgen receptor also can be examined by evaluating the expression of genes whose transcription is regulated by androgen receptor binding. Such genes include PSA, h2k, NKX3.1, and ODC. The amount of transcript and/or protein of such genes in the presence and absence of the compound can be readily determined using art-routine methods such as those described herein. Alternatively, prostate cancer cells in culture can be made transgenic for one or more androgen-regulated genes and the expression of such transgenes can be evaluated in the presence and absence of a compound.

[0052] In addition, the amount of c-jun protein can be evaluated as an indicator of androgen receptor activity. When overexpressed, c-jun has been shown to inhibit the nuclear localization of the androgen receptor. c-jun is a partner with c-fos in the transcription factor AP-1. Increased evidence suggests that the function of the androgen receptor may be affected by an interaction with AP-1.

[0053] Compositions and Articles of Manufacture

[0054] The invention provides compositions that include silymarin or a component thereof or a derivative thereof and at least one other compound selected for its particular mechanism of action on the androgen receptor. The mechanism of action exerted by the other compound(s) can be one or more of the following: inhibition of the expression of a gene encoding an androgen receptor; inhibition of the nuclear localization of an androgen receptor; or inhibition of the nuclear localization of an androgen receptor. Representative compounds exhibiting such mechanisms of action include the following: POH, resveratrol, and omega-3 fatty acids (transactivating ability); flufenamic acid, tea polyphenols (e.g., (−)-epigallocatechin gallate (EGCG)), and quercetin (expression); and numerous anti-androgen compounds (e.g., bicalutamide, flutamide, nilutamide, or cyproterone).

[0055] Compositions containing silymarin or a component thereof can be formulated for delivery to the prostate. In one aspect, silymarin or a component thereof is formulated for transdermal delivery to the prostate. In another aspect, compositions containing silymarin or a component thereof can be formulated for implantation in or near the prostate. Delivery of compositions containing silymarin or a component thereof directly to the prostate of an individual inhibits the nuclear localization of the androgen receptor. Formulations for administration of silymarin or a component thereof described above and apply as well to the disclosed compositions containing silymarin or a component thereof.

[0056] A composition containing silymarin or a component thereof can be in any form provided the composition can be administered to an individual in an amount and for a duration effective to inhibit the nuclear localization of the androgen receptor gene, thereby inhibiting the proliferation of prostate cancer cells. Pharmaceutically acceptable carriers include solvents, dispersion media, coatings, antibacterial and anti-fungal agents, isotonic and absorption delaying agents and the like, appropriate to specific routes of administration.

[0057] Silymarin or a component thereof compositions of the invention that are effective for inhibiting nuclear localization of the androgen receptor as described herein can be combined with packaging material and sold as a kit (i.e., an article of manufacture). Components and methods for producing articles of manufactures are well known. In addition to a composition containing articles of manufacture can include oligonucleotide probes, antibodies, and/or other useful agents for determining the nuclear localization of the androgen receptor. Instructions describing how the composition can be used for inhibiting the nuclear localization of the androgen receptor to thereby inhibit the proliferation of prostate cancer cells can be included in such kits.

[0058] In accordance with the present invention, there may be employed conventional molecular biology, microbiology, biochemical and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES

Example 1

Cell Culture, Cell Proliferation Assays, and PSA and hK2 Quantification Assays

[0059] Human prostate cancer cell line, LNCaP (American Type Culture Collection (ATCC), Manassas, Va.), was grown in RPMI 1640 medium (Mediatech, Herndon, Va.) supplemented with 5% fetal bovine serum (FBS) and 5% CO2 at 37° C. until reaching approximately 50-70% confluence. The media were changed to serum-free RPMI 1640 at 24 hours prior to experiments being performed to deplete undesired steroids. Cells were then treated with 5% charcoal-stripped FBS RPMI 1640 containing silymarin or silibin (all from Sigma (St. Louis, Mo.), dissolved in DMSO) at designated concentrations with or without 1 nM of mibolerone (Mib) (from New England Nuclear (St. Louis, Mo.), dissolved in ethanol), a non-metabolizable synthetic androgen. Equivalent amounts of solvent were added to control cells.

[0060] LNCaP cells were seeded at 4×104/well in 24-well dishes and treated with silymarin or silibin at designated concentrations in the presence of 1 nM Mib. Five days later, cell proliferation was measured by MTS assay kit (Promega, Madison, Wis.), and PSA and hK2 levels in spent media were determined by the Tandem-E PSA kit (Hybritech Inc., San Diego, Calif.) or Mayo's hK2 assay (Zhang et al., 1999, Endocrin., 140:1665-71).

Example 2

Western Blot Analysis

[0061] LNCaP cells were treated with indicated concentrations of silymarin or silibin in the presence of 1 nM Mib for 24 hrs. Cells were harvested and the whole cell lysate and nuclear extract were prepared as described (Zhu et al., 1999, Endocrin., 140:5451-4). Western blotting analysis was performed according to the protocol described (Id). A mouse antibody against human FKBP51 (1:5,000 dilution) (a gift from Dr. David Toft, Mayo Clinic), the human androgen receptor (1:1000 or 1:2000 dilution) (Pharmingen, San Diego, Calif.) or human Sp1 (1:2000 dilution) (Santa Cruz, Santa Cruz, Calif.) was used as the primary antibody. Ponceau S staining was used for monitoring protein loading and transfer efficiency (Mitchell et al., 1999, Cancer Res., 59:5892-5).

Example 3

DNA Constructs

[0062] The 6 Kb PSA promoter and the androgen receptor promoter constructs (pGL3 SV40, PGL3 SV40-3 ARE, PGL3 or PSA promoter/PGL3) have been described (Zhu et al., 1999, Endocrin., 140:5451-4). To make an hk2 androgen responsive element (hk2 ARE) construct, a DNA fragment containing three copies of hk2 ARE (5′-GGAACATATTGTATT-3′ (SEQ ID NO: 1)) was synthesized by the Mayo Molecular Core Facility. The synthesized fragment, including SacI and XhoI restriction enzyme sites at the 5′ and 3+-ends, respectively, was digested with SacI and XhaI according to the manufacturer's instructions, and inserted into a pGL3-Promoter vector (Promega). The fidelity of this construct was confirmed by DNA sequencing.

Example 4

Transient Transfection Assays

[0063] LNCaP cells in duplicate plates were co-transfected with a CMV-β-galactosidase (β-gal) expression vector (0.3 μg/plate) and one of the following: a pGL3-Basic luciferase vector (Promega) containing the PSA promoter (nucleotides 1-5836 of GenBank Accession No. U37672), a pGL3-Promoter luciferase vector (Promega) containing three copies of hk2 ARE, a PGL3 vector or a PSA promoter/pGL3 vector. Transfections were performed using a liposome method with dimethyldioctadecyl-ammonium bromide (Sigma) and L-lecithin (Sigma) (4:10). Cells were then treated with silymarin or silibin in the absence or presence of 1 nM Mib for 24 hrs. Cell extracts were prepared and used for luciferase and β-gal assays (Promega). The β-gal activity was used as a control for transfection efficiency. The above experiments were repeated three times.

Example 5

Ligand Binding Analysis

[0064] LNCaP cells were plated at approximately the same cell number in culture dishes and treated with silymarin or silibin in the presence of 1 nM of 3H-Mib (New England Nuclear) for 24 hrs in the presence or absence of 100 nM cold Mib. Cell pellets were washed twice with 1×phosphate buffered saline (PBS), and incubated with 95% ethanol for 30 min at room temperature. The ethanol-extracted supernatants were then removed for scintillation counting. The difference in radioactivity between groups with and without cold Mib represents the specific ligand-binding activity of androgen receptor. The mean difference from triplicate measurements was determined.

Example 6

Immunocytochemical Staining

[0065] LNCaP cells grown in glass slide chambers (Sigma) were treated with silymarin or silibin at indicated concentrations in the absence or presence of 1 nM Mib. Twenty-four hrs later, LNCaP cell slides were washed with 1×PBS, and fixed in acetone at −20° C. for 10 min. The air-dried slides were treated with EtOH/chloroform (1:1) mixture for 5 min to permeate the cell membrane and air-dried again. The slides were washed with 1×TBST and stained with a monoclonal antibody against the human androgen receptor (Pharmingen) using a DAKO kit (DAKO). The androgen receptor staining was observed using microscopy (Zeiss, Thornwood, N.Y.). The density of androgen receptor in a nucleus (or a whole cell) and the area of the nucleus or the whole cell were quantified by KS400 software (Zeiss) provided by the Mayo Molecular Core Facility. The androgen receptor protein level in a nucleus or a whole cell was calculated by multiplication of the density of androgen receptor in the nucleus or the whole cell with the area of the nucleus or the whole cell, respectively. In each sample, 20 to 25 cells were analyzed. All results were analyzed by 2-tailed Student's t-test. A p<0.05 was accepted as the level of significance.

Example 7

Effect of Silymarin or Silibin on the Androgen Receptor

[0066] LNCAP cell growth increased significantly in the presence of Mib (FIG. 1). Both silymarin or silibin, however, inhibited androgen-stimulated cell growth (FIG. 1). Additionally, neither silymarin or silibin affected the cell proliferation in the absence of Mib (FIG. 1). Silymarin at a concentration of 50 μg/ml or silibin at a concentration of 75 μM fully suppressed the androgen-stimulated cell growth (FIG. 1).

[0067] Prostate-specific kallikrein genes PSA and hK2 are primarily androgen-regulated, and have been used as monitors of androgen receptor action. Their promoters contain androgen-responsive elements (AREs) for androgen receptor binding. As shown in FIGS. 2A and 2B, silymarin or silibin inhibited PSA and hK2 accumulation in LNCaP cells stimulated by androgens.

[0068] FKBP51, an FK506-binding immunophilin, is androgen-regulated and can be detected in functionally mature steroid receptor complexes along with Hsp90 and p23. The FKBP51 protein level was induced by androgens in LNCaP cells. Both the silymarin and silibin treatments reduced the androgen-induced FKBP51 protein level as determined by Western blot analysis.

[0069] Since the androgen receptor is the major regulator of PSA expression, a luciferase reporter gene containing the PSA promoter or a minimal SV40 promoter with 3 copies of hK2 ARE were transfected into LNCaP cells to determine the mechanism by which silymarin or silibin affect androgen-regulated gene expression. Silymarin or silibin significantly reduced the androgenic inducibility of the PSA promoter (FIG. 3A). As shown in FIG. 3B, silymarin or silibin inhibited the ARE-regulated luciferase activity. These results demonstrate that silymarin or silibin inhibit the androgen receptor-mediated transcription activity. Silymarin exerts about twice the inhibitory effect on the androgen receptor as does silibin.

[0070] Therefore, the effect of silymarin or silibin was examined on the expression of the androgen receptor. As shown in FIG. 4A, silymarin or silibin did not affect the androgen-enhanced androgen receptor protein levels in whole cell extracts (p>0.05). Moreover, when a luciferase reporter plasmid containing the androgen receptor promoter was transfected into LNCaP cells, the androgen receptor promoter activity was not affected by silymarin (p>0.05) (FIG. 4B). Thus, these results indicate that the silymarin-mediated inhibition of the androgen receptor function may not be due to an alteration in the total androgen receptor protein level. To exclude the possibility that silymarin or silibin might affect the androgen binding ability of the androgen receptor, a ligand-binding assay was performed. Results demonstrated that silymarin or silibin did not alter the androgen-binding activity of the androgen receptor (p>0.05) (FIG. 4C).

[0071] As a transcription factor, the nuclear level of the androgen receptor is critical for its function. Therefore, the androgen receptor protein level in the nucleus was further investigated. As determined by Western blotting, both silymarin or silibin reduced nuclear androgen receptor levels significantly. Silymarin did not affect the nuclear level of transcription factor Sp1. In addition, and consistent with the above results, silymarin seemed to exert a greater inhibitory effect on nuclear localization (about two-fold) than silibin. Hence, silymarin or silibin reduced the function of the androgen receptor in LNCaP cells by lowering the nuclear androgen receptor level.

[0072] Immunocytochemical staining was performed to further evaluate the alteration of androgen receptor protein levels in whole cells and nuclei. As above, in the absence of androgens, the androgen receptor protein level was low. Mib increased the androgen receptor level significantly, with most of the androgen receptor located in the nuclei. The relative whole cell androgen receptor amount is shown in FIG. 5A, and the ratio of the androgen receptor in nuclei over that in whole cells is shown in FIG. 5B. Consistent with Western blot results, silymarin and silibin did not affect whole cell androgen receptor levels in the presence of Mib (FIG. 5A). However, the nuclear androgen receptor level was reduced significantly by silymarin or silibin treatment (p<0.05) (Figure 5B).

[0073] Silymarin or silibin block the androgen-stimulated proliferation of LNCaP cells. In the presence of androgens, silymarin or silibin inhibited the expression of PSA, hk2 and FKBP51, demonstrating that silymarin or silibin negatively affect the androgen receptor-mediated androgen action. It is also noted that silymarin at a concentration of 50 μg/ml shows a stronger inhibitory effect on the function of the androgen receptor than does an equivalen amount of silibin (75 μM) alone, possibly due to additional flavonoid components in silymarin. Results from experiments described herein demonstrate that silymarin or silibin have a novel action in interfering with the androgen receptor function.

OTHER EMBODIMENTS

[0074] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Methods and compositions for inhibiting the proliferation of prostate cancer cells

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